Kurdistan Regional Government Ministry of Higher Education and Scientific Research Sulaimani University Faculty of Medical Sciences School of Medicine Biochemistry Department
CORRELATION BETWEEN SERUM TRYPTASE LEVELS AND DISEASE SEVERITY IN ASTHMATIC PATIENTS IN SULAIMANI GOVERNORATE
A thesis submitted to the Council of Postgraduate studies of Faculty of Medical Sciences/School of Medicine at University of Sulaimani in partial fulfillment of the requirements for the degree of Masters of Science in Clinical Biochemistry
BY MUHAMMAD AHMED MAHMOOD M.B.CH.B BIOCHEMISTRY DEPARTMENT SUPERVISED BY Dr. BAN MOUSA RASHID (B.Sc. M.Sc. Ph.D. in Clinical Biochemistry)
2015
2715
“Let the future tell the truth and evaluate each one according to his work and accomplishments. The present is theirs; the future, for which I really worked, is mine” -Nikola Tesla
II
I, Muhammad Ahmed Mahmood, declare that this is my original work and has never been presented in any other university and that all resources have been fully acknowledged.
Signature
Muhammad Ahmed Mahmood April 8th. 2015
III
Supervisor Certification I certify that this thesis, entitled “Correlation between Serum Tryptase Levels and Disease Severity in Asthmatic Patients in Sulaimani Governorate”, was prepared under my supervision at the Department of Biochemistry/School of Medicine/Faculty of Medical Sciences/University of Sulaimani in partial fulfillment of the requirements for the degree of Masters of Science in Clinical Biochemistry.
Signature:
Dr. Ban Mousa Rashid PhD in Clinical Biochemistry Date: Apr. 8th 2015
In a view of the available recommendation, I forward this thesis for debate by the examining committee.
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Proofreader: Date: Department of English, School of Language, Faculty of Humanities, University of Sulaimani.
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Examining Committee Certification
We, the Examining Committee after reading this thesis entitled Correlation of serum Tryptase levels to disease severity in asthmatic patients in Sulaimani governorate and examining the student Muhammad Ahmed
Mahmood in its content. It meets the basic requirements for the degree of Masters of Science in Clinical Biochemistry.
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Dedicated To: My dearest wife, Solav; My little angel, Lila; My sweet, Parents; And my Family.
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Acknowledgements I would like to express my sincere gratitude and appreciation to the following people for their concern, encouragement and support, which inspired me to write this thesis. Dr. Ban Mousa Rashid, my supervisor, was an inspiration to me in continuing and finishing my thesis, without whom processing this research could not be possible. She is a wealth of knowledge and renowned expert in her field. Her enthusiasm and interest in my project made me complete my thesis with ease and confidence. My sincere appreciation and special thankfulness to Dr. Beston Faiek Nore for his support and continuous concern throughout my entire work. I would like to thank the following friendly and cooperative doctors at Asthma and Allergy Center, Dr. Jwan Mirza Majeed, Dr. Nzar A. Hama Saeed, Dr. Adil Karim Fatah and Dr. Sherko Ali Omer, and also Paywand Fayaq Jalal, Ahmed Khedir Sofi along with the staff there for letting me do part of my sample collection at their laboratory and offering help whenever I asked. My special gratitude for Dr. Dana Tofiq and Dr. Kosar Muhammad Ali for guiding and easing my work. Many thanks to the Faculty of Medical Sciences, School of Medicine and Department of Biochemistry. I want to express my gratitude to Dr. Hawbir Jamal Nader at Biochemistry Department, and Mr. Alan Ihsan Fawzi and Mr. Emad Hafid Abdul Hamid at Central Laboratory for their great assistance by letting me use their labs at any time. I want to thank Dr. Zhian Salah and Dr. Thaer Eisa Murad for helping me with the statistical work. VIII
My special thanks to my parents for making the person I am today and for their constant support during my study. My special gratitude for my family and friends for giving me unflinching encouragement and their indispensable help in my life and work. Words fail me to express my appreciation to my wife Solav whose dedication, love and persistent confidence in me have taken the load off my shoulder. I owe her for unselfishly letting her intelligence, passion, and ambition collide with mine. Finally, I would like to thank everybody who was important to the successful completion of thesis.
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Abstract Background and objective: Asthma is a chronic inflammatory disorder of the airway that affects more than 300 million individuals worldwide. Tryptase is a neutral serine protease and is the most abundant mediator stored in mast cell granules. The release of tryptase from the secretory granules is a characteristic feature of mast cell degranulation. This study tends to evaluate the difference between serum tryptase levels and asthma severity in Sulaimani governorate.
Methods: Five milliliters of venous blood were obtained from normal and asthmatic subjects (Total n = 145). Eighty- five asthmatic patients were subdivided into three groups according to GINA guideline by the World Health Organization to controlled asthma (n = 25), partly controlled asthma (n = 30) and uncontrolled asthma (n = 30). Serum tryptase level was analyzed using sandwich ELISA.
Results: There was a significant difference among the study groups, with highest serum tryptase levels in uncontrolled asthma (7.68 ± 0.65 ng/dl) and lowest levels in controlled asthma (4.4 ± 0.15 ng/dl). Serum tryptase levels increase significantly with age. There was a significant difference between Ex-smokers and never-smokers in asthmatic subjects. No significance in serum tryptase levels was found between males and females and between asthmatic subjects with negative and positive family history of asthma.
Conclusions: Serum tryptase levels in uncontrolled asthma was significantly higher than controlled asthma. Serum tryptase level is elevated with age and also in ex-smoker asthmatic subjects. However, there was no significant difference in the levels of serum tryptase according to gender and family history of asthma.
X
List of Contents Subject Titles
Page NO.
List of Contents
XII
List of Figures
XVI
List of Tables
XVII
List of Abbreviations
XVIII
Chapter One: Introduction and Literature Review
1
1.1. Asthma
2
1.1.1. Asthma Classification According to GINA Guidelines
2
1.1.2. Diagnosis of Asthma
5
1.1.3. Pathophysiology and Pathogenesis of Asthma
7
1.1.3.1. Bronchoconstriction
7
1.1.3.2. Airway Edema
8
1.1.3.3. Airway Hyperresponsiveness (AHR)
9
1.1.3.4. Airway Remodeling
9
1.1.4. Pathophysiological Mechanism in the Development of
10
Airway Inflammation 1.1.4.1. Inflammatory Cells
10
1.1.4.1.1. Lymphocytes
10
1.1.4.1.2. Basophils
11
1.1.4.1.3. Mast Cells
11
1.1.4.1.4. Eosinophils
14
1.1.4.1.5. Neutrophils
14
1.1.4.1.6. Dendritic Cells
14
1.1.4.1.7. Macrophages
15 XI
1.1.4.1.8. Structural Cells
15
1.1.4.2. Inflammatory Mediators
16
1.1.4.2.1. Chemokines
16
1.1.4.2.2. Cytokines
17
1.1.4.2.3. Cysteinyl-leukotrienes
17
1.1.4.2.4. Nitric Oxide (NO)
17
1.1.4.2.5. Immunoglobulin E (IgE)
18
1.1.4.2.6. Endothelins (ET)
18
1.1.5. Etiology of Asthma
20
1.1.5.1. Host Factors
20
1.1.5.1.1. Atopy
20
1.1.5.1.2. Genetics
21
1.1.5.1.3. Age and Gender
21
1.1.5.1.4. Obesity
22
1.1.5.2. Environmental Factors
22
1.1.5.2.1. Allergens
22
1.1.5.2.2. Infections
23
1.1.5.2.3. Occupational Sensitizers
24
1.1.5.2.4. Environmental Tobacco Smoke (ETS)
24
1.1.5.2.5. Air Pollution
25
1.1.5.2.6. Diet
25
1.2.Tryptase
26
1.2.1. Gene
26
1.2.2. β-Tryptase Structure
27
1.2.3. Tryptase Regulation
28
1.2.4. Biological Activities of β-Tryptase Relevant to Asthma
29
XII
1.3. Aim of the Study
32
Chapter Two: Material and Methods
33
2.1. Study Design
34
2.2. Materials
35
2.3. Methods
36
2.3.1. Sample Collection
36
2.3.2. Norma Value of Serum Tryptase
36
2.3.3. Measurement of Human Mast Cell Tryptase (MCT)
36
2.3.3.1. Principle of the Assay
36
2.3.3.2. Reagents
37
2.3.3.3. Assay Procedure
38
2.3.4. Pulmonary Function Tests (PFT)
39
2.3.5. Statistical Analysis
39
Chapter Three: Results
40
3.1. Serum Tryptase Level and Asthma
41
3.2. Serum Tryptase Levels in all Groups
42
3.3. Multiple Comparisons of Serum Tryptase Levels between
43
Each Group 3.4. Gender Distribution of the Cases
45
3.5. Age Distribution of the Cases
45
3.6. Serum Tryptase and Family History of Asthma
46
3.7. Serum Tryptase Level and Smoking Status in Asthmatic
47
Patients
Chapter Four: Discussion and Conclusion
49
4.1. Control Group
50
4.2. Serum Tryptase Levels in Asthma
50
XIII
4.3. Serum Tryptase Levels and Gender in Asthma
51
4.4. Serum Tryptase Levels and Age
52
4.5. Serum Tryptase Levels and Family History of Asthma
53
4.5. Serum Tryptase Levels and Smoking Status in Asthmatic
53
Patients 4.6. Conclusions
55
4.7. Recommendations
56
REFERENCE
57
Appendix
72
Summary in Arabic Summary in Kurdish
XIV
List of Figures Figure NO.
Title
Page NO.
1.1
The interplay and interaction between airway inflammation, the clinical symptoms and pathophysiology of asthma
7
1.2
Cross-section of a normal airway and a cross-section of an airway during asthma symptoms, which demonstrate the narrowing of the airway in asthmatic patient
8
1.3
IgE binds to mast cells and effectively bind allergen to stimulate release of inflammatory mediators, such as histamine, from mast cells
13
1.4
Airway epithelial cells may play an active role in asthmatic inflammation through the release of many inflammatory mediators, cytokines, chemokines and growth factors
16
1.5
Many cells and mediators are involved in asthma and lead to several effects on the airways
19
1.6
Tetrameric structure of β-Tryptase
28
1.7
Biological activities of Tryptase
30
2.1
Shows Sandwich ELISA with streptavidin-biotin detection
37
XV
List of Tables Table NO.
Title
Page NO.
1.1 1.2 1.3
Asthma classification by the level of control The most important parameters of PFT Risk factors of Asthma, including host factors and environmental exposures Equipment and chemicals Illustration of ELISA procedure Unpaired t-test illustrates the significant difference between all asthmatic patients and normal control with a (P-value < 0.01) ANOVA test shows means, standard deviations, and standard error of mean of Serum tryptase between asthma groups and normal controls with its P-value Tukey’s test multiple comparison test showing means difference (Mean Diff.), 95% Confidence intervals of difference (CI of Diff.) And significance between each group Unpaired t-test which shows no statistical significant between males and females ANOVA test shows means, standard deviations and standard error of mean of serum tryptase between different age groups and its P-value Unpaired t-test shows means, standard deviations and standard error of mean of serum tryptase in negative and positive family history for asthmatic patients T-test shows the means, standard deviations and standard error of mean (SEM) of serum tryptase in none and ex-smokers
4 6 20
2.1 2.2 3.1
3.2
3.3
3.4 3.5
3.6
3.7
XVI
35 38 41
42
44
45 46
47
48
List of Abbreviations AHR……………………………………….Airway Hyper Responsiveness AM…………………………...……………………Alveolar Macrophages ANOVA……………………………….……………Analysis of Variances BALF……………………………..………Broncho Alveolar Lavage Fluid BMI………………………….…………….…………….Body Mass Index CD……………...……………………………….Cluster of Differentiation cDNA…………...…………………………………..complementary DNA CI……………………………………………………...Confidence Interval COPD……………………………Chronic Obstructive Pulmonary Disease EGF……………………………………………Endothelial Growth Factor ELISA…………………………...Enzyme-Linked Immune Sorbent Assay ET…………………………………………...………………...Endothelins ETS……………………………………….Environmental Tobacco Smoke FEV1……………………………Forced Expiratory Volume in one second FcϵRI………………………………………...………………Fc epsilon RI FGF……………………………………………...Fibroblast Growth Factor FGF-2………………………………...…………Fibroblast Growth Factor FVC…………………………………………………Forced Vital Capacity GINA………………….…………………The Global Initiative for Asthma GM-CSF……...…....Granulocyte/Macrophage Colony Stimulating Factor HRP……………………………………...….……Horse radish Peroxidase IFNɣ……………………………...…………………….Interferon Gamma IgE………………………………………...…………...Immunoglobulin E IGF……………………………...……………..Insulin-like Growth Factor IL…………………………...…………………………………..Interleukin iNOS…………………………..…..……..Inducible Nitric Oxide Synthase XVII
LTC4……………………………………….……………Leukotriene C4 LTD4……………………………………………………Leukotriene D4 LTE4…………………………………………………….Leukotriene E4 MCP…………………………………….Monocyte Chemotactic Protein MCT………………………………….………………Mast Cell Tryptase MCTC………………….……….Tryptase and Chymase positive Mast cells MHC…………………….….……….Major Histocompatibility Complex mMCP………………………………………….murine Mast Cell Protease mRNA……………………………………….Messenger Ribonucleic Acid NO…………………………………………………………….Nitric Oxide PARs………………...……………………...Protease Activated Receptors PEF………………………………………………….Peak Expiratory Flow PDGF………………...……….…………..Platelet-Derived Growth Factor PFT……………………………………………..Pulmonary Function Tests PGD2…………………………………………………….Prostaglandin D2 PHM……………………………...………...Peptide Histidine-Methionine PIV…………………………….……………...………Parainfluenza Virus proMMP-3………………………………….Pro-Matrix Metalloprotease-3 RANTES……………Regulated on Activation T-cell Expressed and Secreted RPM…………………………………..……………….Round Per Minute RSV……………….……………….………...Respiratory Syncytial Virus SCF…………………………………..………………….Stem Cell Factor SD………………………………………..…………...Standard Deviation SEM……………………………………………….Standard Error of Mean ST…………………………………………...…………….Serum Tryptase TARC………………..…...Thymus and Activation Regulated Chemokine XVIII
TGF-β………………………………..Transforming Growth Factor-beta Th………………………………………………………….T-helper cells TMT…………………………………………..Transmembrane Tryptase TNF-α……………………………………..Tumor Necrosis Factor alpha VIP……………………………………….. Vasoactive Intestinal Peptide WHO………………………………………...World Health organization
XIX
Chapter One
Introduction and Literature Review
1. Introduction 1.1. Asthma Asthma is defined as a chronic inflammatory disorder of the airways characterized
by
variable
airway
obstruction
and
bronchial
hyperresponsiveness. Airway obstruction can lead to recurrent episodes of wheezing, breathlessness, chest tightness, and coughing, particularly at night or in early morning (National Asthma and Prevention, 2007). Asthma affects more than 300 million individuals worldwide. It is one of the most common chronic diseases and its global incidence continues to increase. Historically, the increase was assumed to occur predominantly in industrial countries, but recent studies show that asthma is now predominant in developing countries (Eder et al., 2006, Masoli et al., 2004).
1.1.1. Asthma Classification According to GINA Guidelines In 1993, the Global Initiative for Asthma (GINA) was formed. Its goals and objectives were described in a 1995 National Heart, Lung, and Blood Institute (NHBLI) and the World Health Organization (WHO) workshop report: Global Strategy for Asthma Management and Prevention (GSAMP). This report was revised in 2002 and its companion documents have been widely distributed and translated into many languages. As the GINA committees expanded their work, the report was updated annually. The first update was posted in October 2003, a second in October 2004 and a third in October 2005, each including the impact of publications from January through December of the previous year (Koshak, 2007). 2
Newly revised document of November 2006 emphasizes asthma control. There is now good evidence that the clinical manifestations of asthma-symptoms, sleep disturbances, limitations of daily activity, impairment of lung function and use of rescue medications-can be controlled with appropriate treatment. The document now recommends a classification of asthma by level of control: Controlled, partly controlled or uncontrolled as shown in table 1.1 (O'Byrne, 2010). Asthma control may be defined in a variety of ways. In lay terms, control may indicate disease prevention, or even cure. However, in asthma, where neither of these are realistic options at present, it refers to control of the manifestations of disease. The aim of treatment should be to achieve and maintain control for prolonged periods with due regard to the safety of treatment, potential for adverse effects, and the cost of treatment required to achieve this goal. Therefore, the assessment of asthma control should include not only control of the clinical manifestations (symptoms, night waking, reliever use, activity limitation and lung function), but also control of the expected future risk to the patient such as exacerbations, accelerated decline in lung function, and side effects of treatment. In general, the achievement of good clinical control of asthma leads to reduced risk of exacerbations. However, certain patients may continue to experience exacerbations in spite of adequate interval control. Smokers are less likely to achieve control and remain at risk of exacerbations. It should be noted that inhaled glucocorticosteroids both improve clinical control and reduce future risk, but some pharmacological agents are more effective in improving features of clinical control, while others are relative more effective at reducing exacerbations (O'Byrne, 2010).
3
Table 1.1: Asthma classification by the level of control (O'Byrne, 2010). Characteristic
Controlled
Partly controlled
Uncontrolled
(All of the (Any measure present in any following)
week)
Daytime
None
More than twice/week
symptoms
(Twice
or
less/week)
Three
or
more
features
of
partly
controlled
asthma
present in any week Limitations
of None
Any
None
Any
activities Nocturnal symptoms/ awakening Need for reliever/ None rescue treatment
(twice
More than twice /week or
less/week) Lung
function Normal
80%
predicted
or
personal best (if known)
(PEF or FEV1) Exacerbations
<
None
One or more/week
One in any week
Previous GINA documents subdivided asthma by severity based on the level of symptoms, airflow limitation, and lung function variability into four categories: Intermittent, Mild Persistent, Moderate Persistent, or Severe Persistent, although this classification was often erroneously 4
applied to patients already on treatment. The main limitation of this previous method of classification of asthma severity was its poor value in predicting what treatment would be required and what a patient’s response to that treatment might be. In view of these limitations, asthma is now by consensus classified on the basis of the intensity of the treatment required to achieve good asthma control. Mild asthma is one that can be wellcontrolled with low-dose inhaled glucocorticosteroids, leukotriene modifiers or cromones. While severe asthma is the one that requires high intensity treatment to maintain good control, or where good control is not achieved despite high intensity treatment as shown in table (O'Byrne, 2010).
1.1.2. Diagnosis of Asthma To establish a diagnosis of asthma, the followings should be determined: Episodic
symptoms
of
airflow
obstruction
or
airway
hyperresponsiveness are present. Airflow obstruction is at least partially reversible. Alternative diagnoses are excluded. Recommended methods to establish the diagnosis are: Detailed medical history. Physical exam focusing on the upper respiratory tract, chest, and skin. Pulmonary Function Tests (PFT) are important tools in the investigation and monitoring of patients with respiratory 5
pathophysiology. They provide important information relating to the large and small airways, the pulmonary parenchyma, the size and integrity of the pulmonary capillary bed. Also assessing reversibility, including in children 5 years of age or older. Reversibility is determined either by an increase in FEV1 (Forced Expiratory Volume in 1 second) of ≥12 percent from baseline or by an increase ≥10 percent of predicted FEV1 after inhalation of a short-acting bronchodilator (Table 1.1). Additional studies as necessary to exclude alternate diagnoses (National Asthma and Prevention, 2007). Table 1.2: The most important parameters of PFT (Kramme et al., 2011).
6
1.1.3. Pathophysiology and Pathogenesis of Asthma A Variety of changes happen in the airway of an asthmatic patient leading to airflow limitation (Figure 1.1). These include:
1.1.3.1. Bronchoconstriction During an asthma episode, the dominant physiological event starts from clinical symptoms by airway narrowing and a subsequent interference with airflow. The smooth muscles in airways contract and produce excess mucus in response to the exposure of variety of stimuli including allergens or irritants as can be seen in figure 1.2 (Maddox and Schwartz, 2002). Allergen-induced acute bronchoconstriction caused by an IgE-dependent release of mediators from mast cells includes histamine, tryptase, leukotrienes, and prostaglandins that directly contract airway smooth muscle (Busse and Lemanske, 2001). The non-IgE-dependent response also involved in mediating the release of mucus from airway cells. Other stimuli can also cause acute airflow obstruction (including exercise and cold air) (Lee and Stevenson, 2011).
Figure 1.1: The interplay and interaction between airway inflammation, the clinical symptoms and pathophysiology of asthma (National Asthma and Prevention, 2007). 7
1.1.3.2. Airway Edema There is a remarkable hyperplasia of the smooth muscle walls of the bronchi and bronchioles with significant thickening of the submucosal basement membranes. All these are associated with an increased deposition of collagen as shown in figure1.2. Mucosal edema is evident throughout the lung tissue often accompanied by sloughing of the mucosal epithelium of the large and small airways. This increase in muscle mass, mucous glands, and tissue edema leads to a thickened airway wall resulting in decreased airway caliber (Fireman, 2003).
Figure 1.2: Cross-section of a normal airway and a cross-section of an airway during asthma symptoms, which demonstrate the narrowing of the airway in asthmatic patient (Jackson et al., 2014).
8
1.1.3.3. Airway Hyperresponsiveness (AHR) Increased airway responsiveness is an exaggerated airway narrowing in response to many stimuli and is the defining characteristic of asthma. The degree of AHR is related to asthma symptoms and the need for treatment. Inflammation of the airways may increase airway responsiveness which thereby allows triggering the airway widening (Barnes, 1996). The mechanisms influencing airway hyperresponsiveness are multiple, including inflammation, dysfunctional neuroregulation, and structural changes. Inflammation appears to be a major factor in determining the degree of airway hyperresponsiveness (Kroegel, 2009).
1.1.3.4. Airway Remodeling Airway remodeling is a collective term that encompasses the alterations in structural cells and tissues in asthmatic patients’ airway, as opposed to the normal individuals’ airway (Elias et al., 1999). Airway remodeling involves an activation of many of the structural cells, with consequent permanent changes in the airway that increase airflow obstruction, airway responsiveness and render the patient less responsive to therapy (Holgate and Polosa, 2006). These structural changes can include thickening of the sub-basement membrane, sub-epithelial fibrosis, airway smooth muscle hypertrophy and hyperplasia, blood vessel proliferation
and
dilation,
and
mucous
gland
hyperplasia
hypersecretion (National Asthma and Prevention, 2007).
9
and
1.1.4. Pathophysiological Mechanism in the Development of Airway Inflammation Airway inflammation has emerged as an important contributor to mechanisms of asthma. Furthermore, the presence of airway inflammation is present even in the absence of severe symptoms (Azzawi et al., 1992). The late asthmatic response is a complex inflammatory process mediated in part by specific leukocyte populations recruited to the airways (Bradley et al., 1991).
1.1.4.1. Inflammatory Cells Many different inflammatory cells are involved in asthma, even the precise role of each cell type is not yet certain as shown in figure 1.5 (Barnes, 1992).
1.1.4.1.1. Lymphocytes Increased numbers of T-lymphocytes are found in the airways mucosa of patients with asthma (Azzawi et al., 1992), or in asthmatics of variable asthma causation including occupational asthma (Bradley et al., 1991, Azzawi et al., 1990, Bentley et al., 1992, Saetta et al., 1992). The majority of lymphocytes bear CD4-receptors whereas CD8-positive cells are more rarely identified, even during exacerbations of asthma (Corrigan et al., 1995). After allergen challenge, there is an increase in bronchial biopsies of asthmatics of activated T-cells and Th2 cytokines (Robinson et al., 1993, Bentley et al., 1993).
10
1.1.4.1.2. Basophils Basophils are circulating granulocytes that respond to allergic stimuli by migrating and accumulating at sites of allergic inflammation (Iliopoulos et al., 1992). It has been reported that levels of basophil cells are increased in the airways of asthmatic as compared with normal subjects (Kimura et al., 1975), and basophil cells are further increased during asthma exacerbations and in response to allergen inhalation challenge (Maruyama et al., 1994, Guo et al., 1994).
1.1.4.1.3. Mast Cells Mast cells have long been deemed to play an important role in the pathophysiology of asthma through their capability to release a host of pleiotropic autacoid mediators, proteases, and cytokines in response to activation by both immunoglobulin E (Kramme et al.)-dependent and diverse non-immunologic stimuli (Bradding et al., 2006). In general with other leukocytes, mast cells develop from CD34+ bone marrow progenitors (Austen and Boyce, 2001, Williams and Galli, 2000, Boyce, 2003). In the airway tissues of asthmatics, mast cells are increased simultaneously with local recruitment and activation of Th2 lymphocytes. Lung mast cells are commonly classed as ‘mucosal’ in opposition to the ‘connective tissue’ mast cells of skin and the peritoneal cavity. These mast cells contain predominantly tryptase and have a scroll-like morphology unlike the grating or lattice like structure of mast cells of the skin (Weidner and Austen, 1990). Two subpopulations of lung mast cells may be involved in the asthmatic response (Forsythe and Ennis, 1998). One population, located beneath the basement membrane close to blood vessels 11
and the fibrous stroma, may be obtained by enzymatic or mechanical dissociation of whole lung tissue and represent what are commonly termed human lung mast cells. The second population of cells is situated between the basement membrane and the epithelium. These cells may be recovered by the bronchoalveolar lavage (BAL). These BAL mast cells may be the first to contact inhaled allergens and thus mediate the initial stage of the asthmatic response, while the human lung cells most likely play a role following the development of chronic disease (Hart, 2001). It is often assumed that chronic mast cell activation in asthma is driven by exposure of the airways to inhaled aeroallergens, resulting in cross-linking of allergen bound to the mast cell high affinity IgE receptor FcϵRI (Cruse et al., 2005). During an allergic response IgE release from B-cells will bind to mast cells, blanketing the plasma membranes of these immune cells as shown in figure 1.3. Half a million IgE molecules coat the surface of mast cells, binding to the high-affinity IgE receptors (FcϵRI) on membranes with the Fc portion (Amin, 2012). A subsequent exposure to the same allergen cross-links the cell-bound IgE and triggers the release of preformed prostaglandins, histamines and cytokines as explained in figure 1.3 (Nakanishi, 2010, Amin et al., 2005). Their principal biological function is increased vascular permeability and recruitment of inflammatory leucocytes as part of both innate and acquired immunity. Mast cell activation stimulates the immediate formation of certain eicosanoids and prostanoids, each again with the immediate goal of vascular changes and recruitment of inflammatory leucocytes (Hart, 2001).
12
Within the first few minutes following laboratory allergen challenge, secretion of the autacoid mediators histamine, prostaglandin D2 (PGD2) and LTC4 induces bronchoconstriction, mucus secretion, and mucosal edema, which account for the acute symptoms as explained in figure 1.3 (Bradding, 2008). However, mast cells also synthesize and secrete a large number of proinflammatory cytokines (including IL-4, IL-5, and IL-13), which regulate both IgE synthesis and the development of eosinophilic inflammation, and several profibrogenic cytokines, including TGF-β and basic fibroblast growth factor (FGF-2) (Bradding and Holgate, 1999). The serine proteases, tryptase, chymase, and carboxy-peptidase are major secretory products of human mast cells that can interact with diverse cell types via protease activated receptors (PARs) and by other processes to alter their behavior greatly (Bradding et al., 2006).
Figure 1.3: IgE binds to mast cells and effectively bind allergen to stimulate release of inflammatory mediators, such as histamine, from mast cells (Amin, 2012).
13
1.1.4.1.4. Eosinophils Increased numbers of eosinophils exist in the airways of most, but not all, persons who have asthma (Chu and Martin, 2001, Sampson, 2000, Williams, 2004). Most allergic and non-allergic asthmatics, including those with mild asthma, have a bronchial eosinophilia and there is a significant association between eosinophil activation and asthma severity (Bousquet et al., 1990). These cells contain inflammatory enzymes, generate leukotrienes, and express a wide variety of pro-inflammatory cytokines.
1.1.4.1.5. Neutrophils Neutrophils are increased in the airways and sputum of persons who have severe asthma, during acute exacerbations (Fahy et al., 1995). The role of neutrophils in stable asthma is unclear. Although recovered in the sputum of asthmatics, neutrophils are usually found in low numbers in BALF (Broncho Alveolar Lavage Fluid), and bronchial biopsies from asthmatic subjects (Fahy et al., 1993, Jeffery et al., 1989, Lacoste et al., 1993, Bradley et al., 1991).
1.1.4.1.6. Dendritic Cells Dendritic cells function as key antigen-presenting cells that interact with allergens from the airway surface and then migrate to draining lymph nodes, suggesting an innate role in initiating immune response against airborne antigen (Murphy and O'Byrne, 2010). Dendritic cells in epithelial cells of asthmatic patients express major histocompatibility complex
14
(MHC). Their role in asthma is still a matter of debate (Lambrecht et al., 1996).
1.1.4.1.7. Macrophages Alveolar macrophages (AM) recovered by BALF have been extensively studied in asthma, and most studies have shown their increased activation and revealed a significant correlation between their activation and the severity of asthma (Godard et al., 1982, Joseph et al., 1983, Chanez et al., 1996, Cluzel et al., 1987, Kelly et al., 1988).
1.1.4.1.8. Structural Cells Structural cells of the airways, including epithelial cells, endothelial cells, fibroblasts and even airway smooth muscle cells may also be a substantial source of inflammatory mediators, like cytokines, lipid mediators in asthma endothelins, chemokines and growth factors as shown in figure 1.4 (Devalia and Davies, 1993, Levine, 1995, Saunders et al., 1997, Johnson and Knox, 1997, Chung, 2000). Epithelial cells may have a crucial role in translating inhaled environmental signals into an airway inflammatory response and are probably the major target cell for inhaled glucocorticoids (Barnes, 1996).
15
Figure 1.4: Airway epithelial cells may play an active role in asthmatic inflammation through the release of many inflammatory mediators, cytokines, chemokines and growth factors (Barnes, 1996).
1.1.4.2. Inflammatory Mediators Many different mediators have been implicated in asthma and they may have a variety of effects on the airways which could account for the pathological features of asthma (Barnes et al., 1998) (Figure 1.5).
1.1.4.2.1. Chemokines A common feature of the response to allergen challenge in asthmatic individuals is pattern of intense inflammation in the airways and a group of chemotactic cytokines, also known as chemokines, are believed to play a central role at multiple stages of this inflammatory response (John and Lukacs, 2003). The discovery of chemokines and the demonstration that some members of this cytokine superfamily are implicated in the 16
recruitment of eosinophils offers an opportunity for a novel therapeutic approach in asthma (Teran, 2000).
1.1.4.2.2. Cytokines Cytokines play an integral role in the coordination and persistence of the inflammatory process in the chronic inflammation of the airways in asthma since they are capable of inducing many of the pro-inflammatory effects that are characteristics of this disease (Chung and Barnes, 1999). Many inflammatory cells (macrophages, mast cells, eosinophils and lymphocytes) are capable of synthesizing and releasing these proteins and structural cells such as epithelial cells. Endothelial cells may also release a variety of cytokines and may therefore participate in the chronic inflammatory response (Barnes, 1994a).
1.1.4.2.3. Cysteinyl-leukotrienes The cysteinyl-leukotrienes LTC4, LTD4 and LTE4 are potent constrictors of human airways and have been reported to increase AHR and may play an important role in asthma (Arm and Lee, 1993). They are the only mediator whose inhibition has been specifically correlated with amelioration in lung function and asthma symptoms (Busse, 1996, Leff, 2001).
1.1.4.2.4. Nitric Oxide (NO) Nitric oxide (NO) is synthesized from the amino acid arginine by enzymes called nitric oxide synthases (Ashutosh, 2000). NO is an intercellular transmitter, both in the central and in the peripheral nervous system. NO plays a pivotal role as a vasodilator, neurotransmitter, and 17
inflammatory mediator in the airways and is produced in increased concentrations in asthma (Kharitonov et al., 1994).
1.1.4.2.5. Immunoglobulin E (IgE) Allergic conditions are almost invariably associated with elevated level of IgE, because IgE is the antibody responsible for activation of allergic reactions and is important to the pathogenesis of allergic diseases. IgE binds to high-affinity receptors on the cell surfaces. The interaction of IgE with antigen is known to lead to a variety of immunological squeal. Cross-linking of IgE bound to mast cells by FcϵRI triggers the release of preformed vasoactive mediators, synthesis of prostaglandins and leukotrienes, and the transcription of cytokines. In the bronchial mucosa, these mediators of immediate-hypersensitivity reactions rapidly induce mucosal edema, mucous production, smooth muscle constriction, and eventually elicit an inflammatory infiltrate (Oettgen and Geha, 1999).
1.1.4.2.6. Endothelins (ET) Endothelins are a family of 21 amino-acid regulatory peptides. ETs are secreted from epithelial, macrophage and endothelial cells (Chanez et al., 1996, Black et al., 1989). Endothelins are potent mediators that are potent vasoconstrictors and bronchoconstrictors (Barnes, 1994b). ETs also cause airway smooth muscle cell proliferation and fibrosis and may subsequently play a role in the chronic inflammation of asthma.
18
Figure 1.5: Many cells and mediators are involved in asthma and lead to several effects on the airways (Longo, 2012).
19
1.1.5. Etiology of Asthma Risk factors for asthma can be divided into those that trigger asthma symptoms and those that cause the development of asthma; some do both as shown in table 1.2. The former are usually environmental factors and the latter include host factors, which are primarily genetic (Busse and Lemanske, 2001). Table 1.3: Risk factors of Asthma, including host factors and environmental exposures (Longo, 2012). Risk Factors of Asthma Host factors: Atopy Genetics Age and Gender Obesity
Environmental factors: Allergens Indoor: Domestic mites, furred animals (dogs, cats and mice), cockroach allergen, fungi, molds and yeasts Outdoor: Pollens, fungi, molds and yeast Infections (predominantly viral) Occupational sensitizers Tobacco smoke Passive smoking Active smoking Air pollution Diet
1.1.5.1. Host Factors 1.1.5.1.1. Atopy Atopy is the major risk factor for asthma, and non-atopic individuals have a very low risk of developing asthma. Patients with asthma commonly suffer from other atopic diseases, particularly allergic rhinitis, which may be found in over 80% of asthmatic patients, and atopic dermatitis (eczema) (Longo, 2012). Atopy, which can be detected by specific serum IgE or 20
skin-test reactivity to environmental allergens, is often associated with asthma. The prevalence of atopy has increased over time in some populations, whereas in others there has been a decrease or a plateau in prevalence since 1990 (Eder et al., 2006).
1.1.5.1.2. Genetics The familial association of asthma and a high degree of concordance for asthma in twins indicate a genetic predisposition to the disease (Longo, 2012). Genetic susceptibility to asthma is due to multiple genes that interact with each other and the environment (Bleecker et al., 1997). Until now, numerous genes have been found that either are implicated in or connected to the existance of asthma and some of its features. The complexity of their involvement in clinical asthma is noted by linkages to certain phenotypic characteristics, but not necessarily the pathophysiologic disease process or clinical picture itself (National Asthma and Prevention, 2007). The search for genes linked to the development of asthma has captured attention on four major areas: IgE production, airway hyperresponsiveness, dysfunctional regulation of the generation of inflammatory mediators (such as cytokines, chemokines, and growth factors) and determination of the ratio between Th1 and Th2 immune response (Strachan, 1989).
1.1.5.1.3. Age and Gender Males have the higher prevalence of asthma in early life. Nonetheless, the sex ratio shifts after puberty and asthma appears predominantly in females (Horwood et al., 1985). Unlike males, females
21
are risk factor for the persistence of asthma in transition from childhood to adulthood (Aberg and Engstrom, 1990, Toelle et al., 2004).
1.1.5.1.4. Obesity Excess weight is identified as a risk factor for the development of asthma, decreased asthma control, increased asthma exacerbations, and increased utilization of emergency services. It may also be responsible for the development of future chronic diseases (Sole, 2013). How asthma is promoted in obesity is still unknown, but it may be a consequence of the combined effects of various factors. Lung function is lower in obese people compared with normal weight people with asthma (Shore, 2008). Adipose tissue’s normal functioning will be increased in obese subjects which in turn leads to a systemic proinflammatory state. Also in obese patients, serum concentrations of numerous cytokines, soluble fractions of their receptors and chemokines have all been found to be abnormal (Delgado et al., 2008). The finding in some studies that there are gender differences in the strength of the relationship between obesity and asthma could suggest that sex hormones play a role modulating this relationship (Beuther et al., 2006).
1.1.5.2. Environmental Factors 1.1.5.2.1. Allergens Despite the fact that indoor and outdoor allergens are well known to cause asthma, it is still not fully resolved. Sensitization and exposure to house hold mite allergens, cat dander, dog dander, and Aspergillus mold are important factors in the development of asthma in children (Sporik and 22
Platts-Mills, 2001). Altough, some recent studies suggest that dog and cat exposure in early life may in fact protect against the development of asthma (Hesselmar et al., 1999). The determinant of these different results has not been established. Studies to evaluate house dust mites and cockroaches exposure have revealed that the prevalence of sensitization and following development of asthma are correlated (Huss et al., 2001, Wahn et al., 1997). The inhalation of a specific allergen by asthmatic subjects can cause three types of airway responses: the isolated early response that develops within 10–30 min after allergen challenge; the isolated late response that develops 3–8 h after the challenge; and the dual response, where subjects develop both early and late airway responses. Furthermore, allergen exposure can promote the persistence of airway inflammation and probability of an exacerbation(Sulakvelidze et al., 1998).
1.1.5.2.2. Infections The role of infections, particularly viral infections, in asthma exacerbations is well-established and their contribution to asthma development and progression increasingly been recognised; however, the relation to asthma severity has rarely been addressed (Chung et al., 2014). Infections with respiratory syncytial virus (RSV) or parainfluenza virus have received much attention because of their tendency to produce a pattern of symptoms termed bronchiolitis, which parallels many of the features of childhood and adult asthma (Gern et al., 1999, Saetta et al., 1992). A number of long-term prospective studies of children admitted to hospital with documented RSV have revealed that about 40 percent of these infants will continue to wheeze or have asthma in later childhood (Sigurs et al., 2000). The “hygiene hypothesis” of asthma suggests that exposure 23
to infections early in life influences the development of a child’s immune system along a “non-allergic” pathway, leading to a reduced risk of asthma and other allergic diseases (Kroegel, 2009).
1.1.5.2.3. Occupational Sensitizers More than 250 substances are known to be correlated with asthma, and the asthma caused by those is known as sensitizer-induced occupational asthma (Lombardo and Balmes, 2000). Initial sensitisation does not usually take place immediately, the process may take from 2 weeks up to 20 or more years to occur. This is called the latency period. Occupational asthma includes: immunoglobulin IgE-mediated asthma after a latency period; irritant asthma with or without a latency period, including reactive airways dysfunction syndrome, which results from high exposure(s); and asthma due to specific occupational agents with unknown pathomechanisms, which also frequently show a latency period (Baur et al., 2012).
1.1.5.2.4. Environmental Tobacco Smoke (ETS) Maternal smoking during pregnancy increases the occurrence of asthma and wheezing during childhood (Gilliland et al., 2001). Furthermore, children with asthma who are exposed to ETS experience more severe symptoms and more frequent exacerbations compared to children without exposure (Halterman et al., 2008). The strong and consistent association between ETS and asthma development in young children may relate to both prenatal and postnatal influences on airway caliber or bronchial responsiveness (Gold, 2000). In adult individuals who have asthma, cigarrete smoking and passive smokers showed increase 24
asthma-related morbidity and disease severity, and also dimineshed responsiveness to inhaled corticosteroids (Stapleton et al., 2011). Cigarette smoking and asthma combine to accelerate the decline in lung function to a greater degree than either factor alone (Thomson et al., 2004).
1.1.5.2.5. Air Pollution Air pollution exposure is associated with increased asthma and allergy morbidity and is a suspected contributor to the increasing prevalence of allergic conditions (Riedl, 2008). Recent studies showed that exposure to air pollution in early life is associated with elevated risks of asthma diagnosis in children (Clark et al., 2010, Ranzi et al., 2014). Environmental exposure to ozone, particulate matter, sulfur dioxide, and nitrogen oxides has been well documented to exacerbate asthma (Tzivian, 2011). Pulmonary function test will also be decreased in children who live in a polluted environment, leading to clinically significant deficits in attained forced expiratory volume at 1 second (FEV1) as children reach adulthood (Gauderman et al., 2004).
1.1.5.2.6. Diet The prevalence of asthma in adults and children is reported to be influenced by many diet-related factors. For example, the antioxidants (vitamins C and E), carotene, riboflavin, and pyridoxin can all exert an important effect by increasing immune function, reducing the symptoms of asthma/eczema, and improving lung function (Delgado et al., 2008).
25
1.2.Tryptase Tryptase is a neutral serine protease with trypsin-like activity. It is one of the major protein components of mast cells in higher eukaryotes (Fiorucci and Ascoli, 2004).
1.2.1. Gene Human tryptases are encoded by at least three genes located together with four or more adjacent tryptase-like pseudogenes on chromosome 16p13.3 (Pallaoro et al., 1999). The tryptases, identified at the cDNA and protein level, are divided into three groups, that is, α, β and γ or transmembrane tryptases (TMTs). So far, eight tryptases (α1, α2; β1a, β1b, β2, β3, γ1, γ2) have been sequenced. Inside each group, members show a high degree of sequence identity (≥98%), indicating that they may be allelic variants of each other. Nevertheless, the number of isoenzymes may be significantly larger as the human tryptase loci are highly polymorphic (Guida et al., 2000). The α/βtryptases encode a 30–amino acid leader and a 245–amino acid catalytic sequence. The α-tryptases show an approximately 90% sequence identity to β-tryptases (Schwartz, 2006). Whereas the γ-TM tryptases (γ-TMT) are less closely related ( ~50% identity with α- and β-tryptases) and contain a C-terminal hydrophobic domain, a feature not found in other tryptases (Sommerhoff, 2001). Also on chromosome 16p13.3 is δ-tryptase(s), originally named murine mast cell protease (mMCP)-7-like tryptase(s) (Pallaoro et al., 1999). The product of this gene reveals close homology to α/β-tryptases over exons 1 through 4, but exon 5 is more closely related to mMCP-7. Even though small amounts may be expressed by mast cells and 26
other cell types, an apparently premature stop codon terminates translation 40 amino acids earlier than α- and β-tryptases (Min et al., 2001). Mast cells appear to express α-, β-, and γ-tryptases. The main types stored in their secretory granules. However, the β-isoenzymes accumulate in much larger amounts than any of the other granule-associated serine proteinases of leukocytes and lymphocytes, comprising as much as 25% of the mast cell protein. For that reason, β-tryptases also are the major isoenzymes that are released during mast cell degranulation and that are isolated from normal human lung and skin tissues (Sommerhoff, 2001).
1.2.2. β-Tryptase Structure Human tryptase is unique in two respects: it is enzymatically active only as a heparin-stabilized tetramer, and it is resistant to all known endogenous proteinase inhibitors. The structure of human beta-tryptase shows four quasi-equivalent monomers. Each monomer contacts its neighbours at two different interfaces through six loop segments (Pereira et al., 1998). The crystal structure reveals that the tetramer is not a compact tetrahedral body as predicted by initial models based on monomeric proteinases. Rather, the monomers (arbitrarily assigned as A, B, C, and D in Figure 1.8) are positioned at the corners of a flat rectangular frame, leaving a continuous central pore. All monomers of the tetramer are nearly equivalent in structure; however, only monomer A is identical to C, and only monomer B is identical to D (Sommerhoff, 2001) (Figure 1.6).
27
Figure 1.6: Tetrameric structure of β-Tryptase (McGrath et al., 2006).
1.2.3. Tryptase Regulation Tryptase is stored in the secretory granules of all human mast cells and is secreted from these cells when they are activated to degranulate (Schwartz et al., 1981). Because β-tryptase is resistant to inhibition by biologic inhibitors of serine proteases such as α1-proteinase inhibitor, α2macroglobulin, and aprotinin, regulation of its activity may depend on regulating its association with heparin proteoglycan (Ren et al., 1998). Tryptase regulation after its release in vivo is uncertain, because the tetrameric enzyme resists inhibition by biologic inhibitors of serine proteases. Regulation may occur, in part, when basic proteins, such as antithrombin III, dissociate the enzyme from heparin (Alter et al., 1990). Purified β-tryptase tetramers in the absence of heparin spontaneously convert to inactive monomers at neutral pH in a physiological salt solution. Nevertheless, the tetrameric structure is stable in high salt solution (>0.5 28
M NaCl). This inactivation, a complex multistep process whose reversibility appears be a function of the pH, is accompanied by conformational changes consistent with the conversion of the active site to a zymogen-like structure (Selwood et al., 1998). The predominant mechanisms regulating the enzymatic activity of tryptase in vivo appears to be the stabilization of the enzymatically active tetramer by aminoglycans and its dissociation into inactive monomers. In contrary to virtually all other serine proteases, human tryptase is resistant to endogenous proteinase inhibitors, resulting in a prolonged catalytic activity in the extracellular space and even in plasma. Tryptase is not inhibited by large proteinase inhibitors like α2-macroglobulin and the serpins (Alter et al., 1990). The mechanism for reconstitution of active tetramer includes conversion of inactive monomers initially to active monomers and then to tetramers (Fukuoka and Schwartz, 2004). At lower concentrations in an acidic pH environment with heparin, active monomers form without progressing to tetramers. Analysis of the initial kinetics of tetramer formation and reactivation suggests a mechanism whereby monomers first form dimers, dimers form inactive tetramers, and inactive tetramers convert to active tetramers (Ren et al., 1998).
1.2.4. Biological Activities of β-Tryptase Relevant to Asthma The biologic activities of enzymatically active tryptase is not clear from the involvement of mast cells in diseases like mastocytosis, anaphylaxis, urticaria, and asthma. The most relevant biologic substrate(s) of tryptase is still uncertain, even though several potential ones have been evaluated, primarily in vitro. β-Tryptase can cleave many proteins. βTryptase rapidly cleaves and inactivates Fibrinogen (Schwartz et al., 1985). 29
Pro-matrix metalloprotease-3 (proMMP-3) is activated by β-tryptase, and it also cleaves low and high molecular weight kininogen (Gruber et al., 1989, Schwartz et al., 1986, Maier et al., 1983). Fibronectin is degraded by β-Tryptase (Lohi et al., 1992). As well as above β-Tryptase activates pro-urokinase and generates C3a from complement C3 (Stack and Johnson, 1994, Schwartz et al., 1983). Vasoactive intestinal peptide (VIP) and calcitonin gene-related peptide are also degraded by β-Tryptase (Caughey et al., 1988, Walls et al., 1992) (Figure 1.7). Apparently, structural features of these proteins allow access to the active sites of the β-Tryptase tetramer (Little and Johnson, 1995).
Figure 1.7: Biological activities of Tryptase. Tryptase augments the contractile potency of other chemical mediators, e.g. histamine; hydrolyses kininogen to produce bradykinin, and cleaves the bronchodilators vasoactive intestinal peptide (VIP) and peptide histidine-methionine (PHM) (Zhang and Timmerman, 1997).
30
Predicted biologic outcomes may include anticoagulation, kinin generation and destruction, fibrosis and fibrolysis, enhancement of vasopermeability, cell surface protease-activated receptor (PAR)-2 activation, angiogenesis, inflammation, and airway smooth-muscle hyperreactivity. Revealing the significance of these potential activities in vivo remains a challenge (Schwartz, 2006). In addition to being a mitogen, tryptase might modulate the migration of cells by cleaving fibrinogen and potentially fibronectin that are enriched in the airways in correlation with injury and inflammation (Thomas et al., 1998). The several proinflammatory actions of tryptase that have been demonstrated in vitro and in vivo involve the capability to cause the accumulation of eosinophils and neutrophils and edema formation (Zhang and Timmerman, 1997).
31
1.3. Aim of the Study The aim of this study is to examine elevation of serum tryptase level and attempt to use it as a biomarker for asthma severity. Thus, this study will evaluate the differnce between serum tryptase levels and disease severity in asthmatic patients in Sulaimani governorate. The project will estimate and include comparisons between serum tryptase levels in asthmatic patients and non-asthmatic individuals.
32
Chapter Two
Material and Methods
II
2.1. Study Design The samples for the study include 145 (72 males and 73 females), including 60 control individuals. All of the recruited individuals gave their informed consent before they were engaged in the study. Eighty-five of the participants are known and already diagnosed asthma cases. The samples collection started from February to August 2014. Sixty samples were collected from healthy controls at the Central Medical Laboratory and eighty-five asthmatic patients mainly at The Center of Asthma and Allergy in Sulaimani Governorate. The Ethics Committee at the School of Medicine, University of Sulaimani, approved the study project. The eighty-five asthmatic patients are divided into three groups according to severity by GINA guidelines (2010) (O'Byrne, 2010). Group 1: Twenty-five samples were patients with controlled asthma. Group 2: Thirty samples were patients with partly controlled asthma. Group 3: Thirty samples were patients with uncontrolled asthma. Inclusion Criteria: 1. Asthmatic patients age more than 5 years 2. Normal control with BMI (Body Mass Index) 18-25 kg/m2 Exclusion criteria: 1. Age less than five years 34
2. Asthmatic patients with other chronic diseases 3. Normal control with BMI more than 25 kg/m2 4. Normal control with chronic diseases
2.2. Materials The equipment, specific reagents and kit used for the study are listed in Table 2.1: Table 2.1: Equipment and chemicals. No.
Equipment and Chemicals
Manufacturer
1.
Micro ELISA reader
BioTek, USA
2.
Centrifuge
Heraeus Labofuge 200, Germany
3.
Incubator
INCD 2, memmert, Germany
4.
Ultra low-temperature freezer (-65 ᵒC) SANYO, Japan
5.
Water Stills/ Distiller
Daihan Labtech, India
6.
Refrigerator (-20 ᵒC)
Vestel, Turkey
7.
Micropipettes (5-50 μl, 20-200 μl, Transferpette, brand, Germany 100-1000 μl)
8.
Water bath
Taeesa, Hanover-Germany
9.
Mast cell tryptase ELISA kit
YH
Bioresearch
laboratory,
Shanghai China 10. Pulmonary Function Test
Jaeger-Toennies, Germany 35
2.3. Methods 2.3.1. Sample Collection Using disposable syringes, five milliliters of venous blood were obtained from participants in the study mainly from arm through Cubital vein. The drawn blood was then stored in a plain tube and left at room temperature for 25 minutes to clot. Then, it was centrifuged for 15 minutes at 3500 rpm to separate the serum and was kept frozen at (-65) ᵒC until they were assayed.
2.3.2. Norma Value of Serum Tryptase Normal range of serum tryptase is established in a commercial test by Thermo Fisher Scientific (ImmunoCap®) that measures serum tryptase level which detects both protryptases and mature forms. The manufacturer has validated the baseline of serum tryptase threshold in individuals without evidence of mast cell stimulation and found a geometric mean of 3.8 ng/dl and 95% upper percentile of 11.4 ng/dl.
2.3.3. Measurement of Human Mast Cell Tryptase (MCT) 2.3.3.1. Principle of the Assay The human mast cell tryptase test uses enzyme-linked immune sorbent assay (ELISA) based on biotin double antibody sandwich technology to assay Human Mast Cell Tryptase (Figure 2.2). MCT is added to each well that is pre-coated with Mast Cell Tryptase monoclonal antibody, and it was then incubated. After incubation, 36
anti MCT antibodies labeled with biotin are added to the unit with streptavidin-HRP, which forms the immune complex. Then, unbound enzymes are removed after washing and then develop using substrate A and B to each well. The solution turns blue and changes to yellow after adding the stop solution. The shades of solution and the concentration of Human Mast Cell Tryptase (MCT) are positively correlated.
Figure 2.1 Shows Sandwich ELISA with streptavidin-biotin detection (Azzopardi et al., 2014).
2.3.3.2. Reagents 1. Coated ELISA plate 12*8 wells 2. Standard solution (48 ng/ml): 0.5 ml 3. Streptavidin-HRP: 6 ml 4. Stop solution: 6 ml 5. Chromogenic reagent A: 6 ml 37
6. Chromogenic reagent B: 6 ml 7. Anti MCT antibodies labeled with biotin: 1 ml 8. Standard dilution: 3 ml 9. Washing concentrate: 20 ml* 30
2.3.3.3. Assay Procedure Table 2.2: Illustration of ELISA procedure Specimen
Blank
Standard
Assay 40 μl
Serum 50 μl
Standard Streptavidin-HRP
50 μl
Anti MCT antibody
10 μl
50 μl
50 μl 10 μl
The plate is covered with seal plate membrane, and shaken gently to mix and then incubating for 60 minute at 37 ᵒC. After that the wells need to be washed with wash solution 5 times. Chromogen reagent A
50 μl
50 μl
50 μl
Chromogen reagent B
50 μl
50 μl
50 μl
Shaking gently to mix and again incubating for 10 more minutes at 37 ᵒC for color development (blue) Stop solution
50 μl
50 μl
Finally the absorbance is measured at 450 nm wavelength
38
50 μl
2.3.4. Pulmonary Function Tests (PFT) The primary instrument used in pulmonary function testing is the spirometer. It is designed to measure changes in volume and can only measure lung volume compartments that exchange gas with the atmosphere. Spirometers with electronic signal outputs (pneumotachs) also measure flow (volume per unit of time). A device is usually attached to the spirometer which measures the movement of gas in and out of the chest and is referred to as a spirograph (Kramme et al., 2011).
2.3.5. Statistical Analysis All the results were expressed as mean ± standard deviation (SD). The data were analyzed by using GraphPad Prism 6.01 software (Graph Pad Software Inc, San Diego, CA, USA). Unpaired t-test and one-way ANOVA followed by Tukey's post hoc test were utilized for statistical evaluation of the differences between the means. P-values< 0.05 were considered to be statistically significant.
39
Chapter Three
Results
II
3.1. Serum Tryptase Level and Asthma The serum tryptase level was measured in this study for asthmatic patients and control individuals. As shown in table 3.1, the results were (4.385 ± 1.248 ng/ml) and (6.025 ± 2.929 ng/ml) in normal control and asthmatic patients respectively. These results clearly indicated significant differences (p <0.05) in the serum tryptase level of control and asthmatic patients. Table 3.1: Unpaired t-test illustrates the significant difference between all asthmatic patients and normal control with a P-value less than 0.05.
Specimen group
No.
Normal Control
60
Serum tryptase (mean ±SD)
4.385 ± 1.248 ng/ml
SEM
P-value
0.161
<0.05
Asthmatic Patients 85
6.025 ± 2.929 ng/ml
41
0.317
3.2. Serum Tryptase Levels in all Groups ANOVA test was carried out for the comparison of serum tryptase levels among asthma groups (controlled, partly controlled and uncontrolled) and normal controls. The results obtained for serum tryptase were (4.3 ± 1.2 ng/ml) in normal control, (4.4 ± 1.5 ng/ml) in controlled asthma group, (5.7 ± 2.2 ng/ml) in partly controlled asthma group and (7.6 ± 3.5 ng/ml) in uncontrolled asthma group. Table 3.2 shows P-value (< 0.05) indicating the significant differences between control and case groups for serum tryptase also confirmed by Tukey’s post hoc test. Table 3.2: A NOVA test shows means, standard deviations, and standard error of mean (SEM) of Serum tryptase between asthma groups and normal controls with its P-value. Serum Specimen group
No. Tryptase
SEM
P-value
(mean ± SD) Normal controls
60
4.3 ± 1.2 ng/ml 0.16
Asthma group: controlled
25
4.4 ± 1.5 ng/ml 0.31 < 0.05
Asthma group: Partly controlled 30
Asthma group: Uncontrolled
30
42
5.7 ± 2.2 ng/ml 0.4
7.6 ± 3.5 ng/ml 0.65
3.3. Multiple Comparisons of Serum Tryptase Levels between Each Group Tukey’s multiple comparisons test was done to find significance between study groups. The results of table 3.3 indicated that there are, no significant differences between normal and controlled asthma group, significant differences between normal and partly controlled asthma group, very highly significant differences between normal and uncontrolled asthma group, no significant differences between controlled asthma group and partly controlled asthma group, very highly significant differences between controlled asthma group and uncontrolled asthma group and highly significant differences between partly controlled asthma group and uncontrolled asthma group.
43
Table 3.3: Tukey’s test multiple comparison test showing means difference (Mean Diff.), 95% Confidence intervals of difference (CI of Diff.) And significance between each group.
Tukey's multiple comparisons test
Mean Diff.
95% CI of Diff.
Significance
Normal vs. Controlled Asthma group
-0.026
-1.36 to 1.31
No
Normal vs. Partly controlled Asthma group
-1.3
-2.59 to -0.07
Yes
Normal vs. Uncontrolled Asthma group
-3.2
-4.55 to -2.03
Very highly
Controlled Asthma group vs. Partly controlled Asthma group
-1.3
-2.83 to 0.22
No
Controlled Asthma group vs. Uncontrolled Asthma group
-3.2
-4.79 to -1.74
Very highly
Partly controlled Asthma group vs. Uncontrolled Asthma group
-1.9
-3.42 to -0.51
Highly
44
3.4. Gender Distribution of the Cases The total number of blood samples from Asthma patients was 85. The number was 42 males and 43 females, as shown in table 3.4. There is no statistically significant difference between serum tryptase level and gender unpaired t-test and the P-value is more than 0.05. Table 3.4: Unpaired t-test which shows no statistical significance between males and females.
Gender
Male
Female
No.
42 43
Serum Tryptase (Mean ± SD)
6.09 ± 2.9 ng/ml 5.9 ± 2.9 ng/ml
SEM
t-value
P-value
0.19
0.8
0.46 0.44
3.5. Age Distribution of the Cases The age distribution of 85 asthmatic patients. Their ages ranged from 5-24, 25-44 and over 45 years. ANOVA and post hoc test were used for different age groups and the result shows that P-value is less than 0.05 as shown in table 3.5, which in turn means that serum tryptase level increases with age. Linear regression also showed a positive correlation between age and serum tryptase levels with R2 = 0.114.
45
Table 3.5: ANOVA test shows means, standard deviations and standard error of mean (SEM) of serum tryptase between different age groups and its P-value.
Serum Tryptase
Age classes
No.
5-24 years
25
5.08 ± 1.7 ng/ml
0.35
25-44 years
26
5.3 ± 1.8 ng/ml
0.37
45 years and above
34
7.2 ± 3.7 ng/ml
0.64
( mean ± SD)
SEM
P-value
<0.05
3.6. Serum Tryptase and Family History of Asthma Of the 85 cases, the asthmatic patients with negative family history were 48 and asthmatic patients with positive family history were 37. As shown in table 3.6, there is no significant difference in tryptase levels between the asthmatic patients with negative family history and asthmatic patients with positive family history.
46
Table 3.6: Unpaired t-test shows means, standard deviations (SD) and standard error of mean (SEM) of serum tryptase in negative and positive family history for asthmatic patients.
Family History
NO
No.
48
Serum Tryptase (means ± SD)
6.498 ± 3.146 ng/ml
SEM
P-value
0.4541 0.09
YES
37
5.412 ± 2.532 ng/ml
0.4163
3.7. Serum Tryptase Level and Smoking Status in Asthmatic Patients Smoking status is divided into two groups: those patients whom never smoked (71 cases) and those that are ex-smokers (14 cases), since there is no any current smokers among participants. As shown in table 3.7 there was a significant difference in serum tryptase levels between neversmokers and ex-smokers of asthmatic patients.
47
Table 3.7: T-test shows the means, standard deviations (SD) and standard error of mean (SEM) of serum tryptase in none and ex-smokers.
Smoking Status No.
Never-smokers
71
Serum Tryptase (mean ± SD)
5.66 ± 2.62 ng/ml
SEM
P-value
0.311 <0.05
Ex-smokers
14
7.86 ± 3.73 ng/ml
48
0.999
Chapter Four
Discussion and Conclusion
II
4.1. Control Group Samples were taken from healthy individuals whose age ranged from 5 to 80 years old. The mean value of serum tryptase ± S.D. was (4.3 ± 1.2 ng/ml) (Table 3.1).
4.2. Serum Tryptase Levels in Asthma Serum tryptase was chosen for the present study because it is a prominent constituent of, and specific to, the mast cell and is relatively more stable than histamine in serum. It is considered to be a reliable indicator of mast cell degranulation (Schwartz et al., 1989). Tryptase levels are increased in the lungs of asthmatics and tryptase inhibitors have been shown to have important effects in reducing both early- and late-phase airway responses (Welle, 1997, Bingham and Austen, 2000, Schwartz, 1992). In the present study asthma severity (classified according to GINA guidelines by WHO) is found significant difference in serum tryptase levels and also a significant difference was confirmed between the levels of asthma severity and normal population (P < 0.05) (Table 3.1 and 3.3). Thus, implicating that serum tryptase levels increase with an increase in disease severity from controlled to partly controlled and uncontrolled asthma in Sulaimani governorate. Similar studies conducted shows that severe asthma is associated with a predominance of tryptase and chymase positive mast cells (MCTC) with increased expression of FcεRI and surface-bound IgE in the airway submucosa and epithelium (Balzar et al., 2011, Andersson et al., 2011). Therefore, the activity of mast cells increases with the increase in the severity of asthma, which is mainly caused by an ongoing inflammation in
50
the alveolar tissue leading to elevated levels of serum tryptase in asthmatic patients.
4.3. Serum Tryptase Levels and Gender in Asthma Among 85 samples in this study, 42 were males and 43 were females. The result shows no statistical difference in serum tryptase levels between males and females (P value > 0.5), but the mean of serum tryptase levels in male subjects were slightly higher than in female subjects (Table 3.4). Correlation between gender and basal serum tryptase levels is still argumentative and controversial. Despite the fact that there is no significant difference between males and females basal tryptase levels, some studies show that males have higher basal tryptase levels than females (Sahiner et al., 2014, Gonzalez-Quintela et al., 2010, Fenger et al., 2012, Komarow et al., 2009, Schliemann et al., 2012). Nevertheless, other studies show that the mean of basal tryptase level in female subjects is slightly higher than in male subjects (Schwartz et al., 2003, Min et al., 2004). The influence of gender could be on the total body burden of mast cells, the production and processing of tryptase by mast cells, or the metabolism of spontaneously secreted tryptase (Min et al., 2004). The effect of gender on the clinical use of total tryptase levels in serum remains to be fully assessed but should be considered when interpreting test results.
51
4.4. Serum Tryptase Levels and Age All participants in this study were divided into three age classes: 524 years, 25-44 years and 45 years and above. The result shows a significant difference between serum tryptase levels and age (P value < 0.01). Therefore, the data reveal that serum tryptase level increases with age (Table 3.5). Although it is well known that serum tryptase level correlate positively with age (Schliemann et al., 2012, Gonzalez-Quintela et al., 2010, Blum et al., 2011, Kucharewicz et al., 2007). A study by Sahiner (2014) shows that in pediatric age group higher level of serum tryptase can be found during infancy age 0-1 year, it also shows that basal tryptase levels in children is similar to those in adults (Sahiner et al., 2014). A study by Tsukioka (2010) indicates that asthma severity is also significantly associated with age at onset in patients with adult-onset asthma (Tsukioka et al., 2010). Thus, it means that serum tryptase level can be used as a good marker for asthma and it can be considered as a risk factor for asthma severity in older individuals.
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4.5. Serum Tryptase Levels and Family History of Asthma Family history is introduced as a strongest risk factor for asthma in adults and childhood asthma (Liu et al., 2009, Mielck et al., 1996, Burke et al., 2003, Subbarao et al., 2009, Kaiser, 2004). Moreover, studies confirmed that a greater asthma severity is associated with a positive family history of asthma (Higgins et al., 2005, Mielck et al., 1996, Ratageri et al., 2000). Among 85 cases of asthma involved in the study, 48 cases had a negative family history, whereas 37 cases were found to have a positive family history with either first or second degree relatives. The result demonstrates no significant difference between family history and serum tryptase levels (Table 3.6). This outcome is also confirmed by Gasiorowska (2006) clarifying that family history of allergy does not correlate with serum tryptase concentrations (Gasiorowska et al., 2006).
4.5. Serum Tryptase Levels and Smoking Status in Asthmatic Patients Cigarette smoking is associated with accelerated decline of lung function, increased mortality, and worsening of symptoms in asthma. Moreover, exposure to cigarette smoke can alter the inflammatory mechanisms in asthma to become similar to that seen in COPD with increasing CD8 cells and neutrophils and may therefore alter the response to therapy. Cigarette smoke exposure has been associated with a poor response to inhaled corticosteroids which are recommended as first line anti-inflammatory medications in asthma (Tamimi et al., 2012).
53
A significant difference is found between serum tryptase levels and smoking status (Table 3.7). The result shows a significant difference between never-smokers and ex-smokers asthmatic patients with a tryptase levels mean higher in ex-smokers than never-smokers, since there is no current smokers in the study individuals. A study by Small-Howard (2005) shows that cigarette smoking upregulates mast cell-secreted proteinases levels in mast cells at both the protein and the mRNA level (Small-Howard and Turner, 2005). Another study by Kalenderian (1988) demonstrates that smokers have elevated histamine and tryptase levels in bronchoalveolar lavage fluid, suggesting increased mast cells degranulation (Kalenderian et al., 1988). Ex-smokers shows persisting mucosal inflammation similar to that seen in current smokers (Gamble et al., 2007, Wen et al., 2010). Therefore, it is expected to observe a higher serum tryptase levels in ex-smokers than never-smokers in asthmatic patients. Furthermore, this study is suggesting that smoking history is preferable to be taken into consideration before measuring serum tryptase level in asthmatic patients.
54
4.6. Conclusions 1. Serum tryptase level is significantly increased with asthma severity. Meaning, serum levels of tryptase are higher in uncontrolled and partly controlled asthma than controlled asthma and normal populations. 2. There is no significant difference in serum tryptase levels in males and females. 3. Older patients showed significantly higher serum tryptase levels. 4. Family history of asthma does not affect serum tryptase concentration in asthmatic patients. 5. Ex-smokers are having higher serum tryptase level. Thus, smoking directly influence tryptase concentration as it does activate mast cells and induce more inflammation.
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4.7. Recommendations 1. Increasing population size to get more reliable and accurate results. 2. Conducting a study to investigate the disease response to anti-tryptase monoclonal antibodies in different asthma classes (controlled, partly controlled and uncontrolled) and to study the effects of anti-tryptase antibodies on the airway inflammation and the levels of serum tryptase in different asthma classes.
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REFERENCE
II
References ABERG, N. & ENGSTROM, I. 1990. Natural history of allergic diseases in children. Acta Paediatr Scand, 79, 206-11. ALTER, S. C., KRAMPS, J. A., JANOFF, A. & SCHWARTZ, L. B. 1990. Interactions of human mast cell tryptase with biological protease inhibitors .Arch Biochem Biophys, 276, 26-31. AMIN, K. 2012. The role of mast cells in allergic inflammation. Respir Med, 106, 9-14. AMIN, K., JANSON, C., BOMAN, G. & VENGE, P. 2005. The extracellular deposition of mast cell products is increased in hypertrophic airways smooth muscles in allergic asthma but not in nonallergic asthma. Allergy, 60, 1241-7. ANDERSSON, C. K., BERGQVIST, A., MORI, M., MAUAD, T., BJERMER, L. & ERJEFALT, J. S. 2011. Mast cell-associated alveolar inflammation in patients with atopic uncontrolled asthma. J Allergy Clin Immunol, 127, 905-12 e1-7. ARM, J. P. & LEE, T. H. 1993. Sulphidopeptide leukotrienes in asthma. Clin Sci, 84, 501-10. ASHUTOSH, K. 2000. Nitric oxide and asthma: a review. Curr Opin Pulm Med, 6, 21-5. AUSTEN, K. F. & BOYCE, J. A. 2001. Mast cell lineage development and phenotypic regulation. Leuk Res, 25, 511-8. AZZAWI, M., BRADLEY, B., JEFFERY, P. K., FREW, A. J., WARDLAW, A. J., KNOWLES, G., ASSOUFI, B., COLLINS, J. V., DURHAM, S. & KAY, A. B. 1990. Identification of activated T lymphocytes and eosinophils in bronchial biopsies in stable atopic asthma. Am Rev Respir Dis, 142, 1407-13. AZZAWI, M., JOHNSTON, P. W., MAJUMDAR, S., KAY, A. B. & JEFFERY, P. K. 1992. T lymphocytes and activated eosinophils in airway mucosa in fatal asthma and cystic fibrosis. Am Rev Respir Dis, 145, 1477-82. AZZOPARDI, L., THOMPSON, S. A., HARDING, K. E., COSSBURN, M., ROBERTSON, N., COMPSTON, A., COLES, A. J. & JONES, J. L. 2014. Predicting autoimmunity after alemtuzumab treatment of multiple sclerosis. J Neurol Neurosurg Psychiatry, 85, 795-8. BALZAR, S., FAJT, M. L., COMHAIR, S. A., ERZURUM, S. C., BLEECKER, E., BUSSE, W. W., CASTRO, M., GASTON, B., ISRAEL, E., SCHWARTZ, L. 58
B., CURRAN-EVERETT, D., MOORE, C. G. & WENZEL, S. E. 2011. Mast cell phenotype, location, and activation in severe asthma. Data from the Severe Asthma Research Program. Am J Respir Crit Care Med, 183, 299-309. BARNES, P. J. 1992. New aspects of asthma. J Intern Med, 231, 453-61. BARNES, P. J. 1994a. Cytokines as mediators of chronic asthma. Am J Respir Crit Care Med, 150, S42-9. BARNES, P. J. 1994b. Endothelins and pulmonary diseases. J Appl Physiol (1985), 77, 1051-9. BARNES, P. J. 1996. Pathophysiology of asthma. Br J Clin Pharmacol, 42, 3-10. BARNES, P. J., CHUNG ,K. F. & PAGE, C. P. 1998. Inflammatory mediators of asthma: an update. Pharmacol Rev, 50, 515-96. BAUR, X., SIGSGAARD, T., AASEN, T. B., BURGE, P. S., HEEDERIK, D., HENNEBERGER, P., MAESTRELLI, P., ROOYACKERS, J., SCHLUNSSEN, V., VANDENPLAS, O., WILKEN, D & .ASTHMA, E. R. S. T. F. O. T. M. O. W.-R. 2012. Guidelines for the management of work-related asthma. Eur Respir J, 39, 529-45. BENTLEY, A. M., MAESTRELLI, P., SAETTA, M., FABBRI, L. M., ROBINSON, D. S., BRADLEY, B. L., JEFFERY, P. K., DURHAM, S. R & .KAY, A. B. 1992. Activated T-lymphocytes and eosinophils in the bronchial mucosa in isocyanate-induced asthma. J Allergy Clin Immunol, 89, 821-9. BENTLEY, A. M., MENG, Q., ROBINSON, D. S., HAMID, Q., KAY, A. B. & DURHAM, S. R. 1993. Increases in activated T lymphocytes, eosinophils, and cytokine mRNA expression for interleukin-5 and granulocyte/macrophage colony-stimulating factor in bronchial biopsies after allergen inhalation challenge in atopic asthmatics. Am J Respir Cell Mol Biol, 8, 35-42. BEUTHER ,D. A., WEISS, S. T. & SUTHERLAND, E. R. 2006. Obesity and asthma. Am J Respir Crit Care Med, 174, 112-9. BINGHAM, C. O., 3RD & AUSTEN, K. F. 2000. Mast-cell responses in the development of asthma. J Allergy Clin Immunol, 105, S527-34. BLACK, P. N., GHATEI ,M. A., TAKAHASHI, K., BRETHERTON-WATT, D., KRAUSZ, T., DOLLERY, C. T. & BLOOM, S. R. 1989. Formation of endothelin by cultured airway epithelial cells. FEBS Lett, 255, 12932.
59
BLEECKER, E. R., POSTMA, D. S. & MEYERS, D. A. 1997. Evidence for multiple genetic susceptibility loci for asthma. Am J Respir Crit Care Med, 156, S113-6. BLUM, S., GUNZINGER, A., MULLER, U. R. & HELBLING, A. 2011. Influence of total and specific IgE, serum tryptase, and age on severity of allergic reactions to Hymenoptera stings .Allergy, 66, 222-8. BOUSQUET, J., CHANEZ, P., LACOSTE, J. Y., BARNEON, G., GHAVANIAN, N., ENANDER, I., VENGE, P., AHLSTEDT, S., SIMONY-LAFONTAINE, J., GODARD, P. & ET AL. 1990. Eosinophilic inflammation in asthma. N Engl J Med, 323, 1033-9. BOYCE, J. A .2003 .Mast cells: beyond IgE. J Allergy Clin Immunol, 111, 24-32; quiz 33. BRADDING, P. 2008. Asthma: eosinophil disease, mast cell disease, or both? Allergy Asthma Clin Immunol, 4, 84-90. BRADDING, P. & HOLGATE, S. T. 1999. Immunopathology and human mast cell cytokines. Crit Rev Oncol Hematol, 31, 119-33. BRADDING, P., WALLS, A. F. & HOLGATE, S. T. 2006. The role of the mast cell in the pathophysiology of asthma. J Allergy Clin Immunol, 117, 1277-84. BRADLEY, B. L., AZZAWI, M., JACOBSON, M., ASSOUFI, B ,.COLLINS, J. V., IRANI, A. M., SCHWARTZ, L. B., DURHAM, S. R., JEFFERY, P. K. & KAY, A. B. 1991. Eosinophils, T-lymphocytes, mast cells, neutrophils, and macrophages in bronchial biopsy specimens from atopic subjects with asthma: comparison with biopsy specimens from atopic subjects without asthma and normal control subjects and relationship to bronchial hyperresponsiveness. J Allergy Clin Immunol, 88, 661-74. BURKE, W., FESINMEYER, M., REED, K., HAMPSON, L. & CARLSTEN, C. 2003. Family history as a predictor of asthma risk. Am J Prev Med, 24, 160-9. BUSSE, W. W. 1996. The role of leukotrienes in asthma and allergic rhinitis. Clin Exp Allergy, 26, 868-79. BUSSE, W. W. & LEMANSKE, R. F., JR. 2001. Asthma. N Engl J Med, 344, 350-62. CAUGHEY, G. H., LEIDIG, F ,.VIRO, N. F. & NADEL, J. A. 1988. Substance P and vasoactive intestinal peptide degradation by mast cell tryptase and chymase. J Pharmacol Exp Ther, 244, 133-7.
60
CHANEZ, P., VIGNOLA, A. M., ALBAT, B., SPRINGALL, D. R., POLAK, J. M., GODARD, P. & BOUSQUET ,J. 1996. Involvement of endothelin in mononuclear phagocyte inflammation in asthma. J Allergy Clin Immunol, 98, 412-20. CHU, H. W. & MARTIN, R. J. 2001. Are eosinophils still important in asthma? Clin Exp Allergy, 31, 525-8. CHUNG, K. F. 2000. Airway smooth muscle cells: contributing to and regulating airway mucosal inflammation? Eur Respir J, 15, 961-8. CHUNG, K. F. & BARNES, P. J. 1999. Cytokines in asthma. Thorax, 54, 825-57. CHUNG, K. F., WENZEL, S. E., BROZEK, J. L., BUSH, A., CASTRO, M., STERK, P .J., ADCOCK, I. M., BATEMAN, E. D., BEL, E. H., BLEECKER, E. R., BOULET, L. P., BRIGHTLING, C., CHANEZ, P., DAHLEN, S. E., DJUKANOVIC, R., FREY, U., GAGA, M., GIBSON, P., HAMID, Q., JAJOUR, N. N., MAUAD, T., SORKNESS, R. L. & TEAGUE, W. G. 2014. International ERS/ATS guidelines on definition, evaluation and treatment of severe asthma. Eur Respir J, 43, 343-73. CLARK, N. A., DEMERS, P. A., KARR, C. J., KOEHOORN, M., LENCAR, C., TAMBURIC, L. & BRAUER, M. 2010. Effect of early life exposure to air pollution on development of childhood asthma. Environ Health Perspect, 118, 284-90. CLUZEL, M., DAMON, M., CHANEZ, P., BOUSQUET, J., CRASTES DE PAULET, A., MICHEL, F. B. & GODARD, P. 1987. Enhanced alveolar cell luminol-dependent chemiluminescence in asthma. J Allergy Clin Immunol, 80, 195-201. CORRIGAN, C. J., HAMID, Q., NORTH, J., BARKANS, J., MOQBEL, R., DURHAM, S., GEMOU-ENGESAETH, V. & KAY, A. B. 1995. Peripheral blood CD4 but not CD8 t-lymphocytes in patients with exacerbation of asthma transcribe and translate messenger RNA encoding cytokines which prolong eosinophil survival in the context of a Th2-type pattern: effect of glucocorticoid therapy. Am J Respir Cell Mol Biol, 12, 567-78. CRUSE, G., KAUR, D., YANG, W., DUFFY, S. M., BRIGHTLING, C. E. & BRADDING ,P. 2005. Activation of human lung mast cells by monomeric immunoglobulin E. Eur Respir J, 25, 858-63. DELGADO, J., BARRANCO, P. & QUIRCE, S. 2008. Obesity and asthma. J Investig Allergol Clin Immunol, 18, 420-5.
61
DEVALIA, J. L. & DAVIES, R. J. 1993. Airway epithelial cells and mediators of inflammation. Respir Med, 87, 405-8. EDER, W., EGE, M. J. & VON MUTIUS, E. 2006. The asthma epidemic. N Engl J Med, 355, 2226-35. ELIAS, J. A., ZHU, Z., CHUPP, G. & HOMER, R. J. 1999. Airway remodeling in asthma. J Clin Invest, 104, 1001-6. FAHY, J. V., KIM, K. W., LIU, J. & BOUSHEY, H. A. 1995. Prominent neutrophilic inflammation in sputum from subjects with asthma exacerbation. J Allergy Clin Immunol, 95, 843-52. FAHY, J. V., LIU, J., WONG, H. & BOUSHEY, H. A. 1993. Cellular and biochemical analysis of induced sputum from asthmatic and from healthy subjects. Am Rev Respir Dis, 147, 1126-31. FENGER, R. V., LINNEBERG, A., VIDAL, C., VIZCAINO, L., HUSEMOEN, L. L., AADAHL, M. & GONZALEZ-QUINTELA, A. 2012. Determinants of serum tryptase in a general population: the relationship of serum tryptase to obesity and asthma. Int Arch Allergy Immunol, 157, 151-8. FIORUCCI, L. & ASCOLI, F. 2004. Mast cell tryptase, a still enigmatic enzyme. Cell Mol Life Sci, 61, 1278-95. FIREMAN ,P. 2003. Understanding asthma pathophysiology. Allergy Asthma Proc, 24, 79-83. FORSYTHE, P. & ENNIS, M. 1998. Pharmacological regulation of mediator release from human mast cells. Clin Exp Allergy, 28, 1171-3. FUKUOKA, Y. & SCHWARTZ, L. B. 2004. Human beta-tryptase: detection and characterization of the active monomer and prevention of tetramer reconstitution by protease inhibitors. Biochemistry, 43, 10757-64. GAMBLE, E., GROOTENDORST, D. C., HATTOTUWA, K., O'SHAUGHNESSY, T., RAM, F. S., QIU, Y., ZHU, J ,.VIGNOLA, A. M., KROEGEL, C., MORELL, F., PAVORD, I. D., RABE, K. F., JEFFERY, P. K. & BARNES, N. C. 2007. Airway mucosal inflammation in COPD is similar in smokers and ex-smokers: a pooled analysis. Eur Respir J, 30, 46771. GASIOROWSKA, J., CZERWIONKA-SZAFLARSKA, M., SWINCOW, G., GRUSZKA, M. & ODROWAZ-SYPNIEWSKA, G. 2006. [Analysis of tryptase levels in infants and children with wheezy bronchitis]. Pol Merkur Lekarski, 21, 516-21.
62
GAUDERMAN, W. J., AVOL, E., GILLILAND, F., VORA, H., THOMAS, D., BERHANE ,K., MCCONNELL, R., KUENZLI, N., LURMANN, F., RAPPAPORT, E., MARGOLIS, H., BATES, D. & PETERS, J. 2004. The effect of air pollution on lung development from 10 to 18 years of age. N Engl J Med, 351, 1057-67. GERN, J. E., LEMANSKE, R. F., JR. & BUSSE, W. W .1999 .Early life origins of asthma. J Clin Invest, 104, 837-43. GILLILAND, F. D., LI, Y. F. & PETERS, J. M. 2001. Effects of maternal smoking during pregnancy and environmental tobacco smoke on asthma and wheezing in children. Am J Respir Crit Care Med ,163 , .36-429 GODARD, P., CHAINTREUIL, J., DAMON, M., COUPE, M., FLANDRE, O., CRASTES DE PAULET, A. & MICHEL, F. B. 1982. Functional assessment of alveolar macrophages: comparison of cells from asthmatics and normal subjects. J Allergy Clin Immunol, 70, 8.93-8 GOLD, D. R. 2000. Environmental tobacco smoke, indoor allergens, and childhood asthma. Environ Health Perspect, 108 Suppl 4, 643-51. GONZALEZ-QUINTELA, A., VIZCAINO, L., GUDE, F., REY, J., MEIJIDE, L., FERNANDEZ-MERINO, C., LINNEBERG, A. & VIDAL, C .2010 .Factors influencing serum total tryptase concentrations in a general adult population. Clin Chem Lab Med, 48, 701-6. GRUBER, B. L., MARCHESE, M. J., SUZUKI, K., SCHWARTZ, L. B., OKADA, Y., NAGASE, H. & RAMAMURTHY, N. S. 1989. Synovial procollagenase activation by human mast cell tryptase dependence upon matrix metalloproteinase 3 activation. J Clin Invest, 84, 1657-62. GUIDA, M., RIEDY, M., LEE, D. & HALL, J. 2000. Characterization of two highly polymorphic human tryptase loci and comparison with a newly discovered monkey tryptase ortholog. Pharmacogenetics, 10, 389-96. GUO, C. B., LIU, M. C., GALLI, S. J., BOCHNER, B. S., KAGEY-SOBOTKA, A. & LICHTENSTEIN, L. M. 1994. Identification of IgE-bearing cells in the late-phase response to antigen in the lung as basophils. Am J Respir Cell Mol Biol, 10, 384-90. HALTERMAN, J. S., BORRELLI, B., TREMBLAY, P., CONN, K. M., FAGNANO, M., MONTES, G. & HERNANDEZ, T. 2008. Screening for environmental tobacco smoke exposure among inner-city children with asthma. Pediatrics, 122, 1277-83. 63
HART, P. H. 2001. Regulation of the inflammatory response in asthma by mast cell products. Immunol Cell Biol, 79, 149-53. HESSELMAR, B., ABERG, N., ABERG, B., ERIKSSON, B. & BJORKSTEN, B. 1999. Does early exposure to cat or dog protect against later allergy development? Clin Exp Allergy, 29, 611-7. HIGGINS, P. S., WAKEFIELD, D. & CLOUTIER, M. M. 2005. Risk factors for asthma and asthma severity in nonurban children in Connecticut. Chest, 128, 3846-53. HOLGATE, S. T. & POLOSA, R. 20 .06The mechanisms, diagnosis, and management of severe asthma in adults. Lancet, 368, 780-93. HORWOOD, L. J., FERGUSSON, D. M. & SHANNON, F. T. 1985. Social and familial factors in the development of early childhood asthma. Pediatrics, 75, 859-68. HUSS ,K., ADKINSON, N. F., JR., EGGLESTON, P. A., DAWSON, C., VAN NATTA, M. L. & HAMILTON, R. G. 2001. House dust mite and cockroach exposure are strong risk factors for positive allergy skin test responses in the Childhood Asthma Management Program. J Allergy Clin Immunol, 107, 48-54. ILIOPOULOS, O., BAROODY, F. M., NACLERIO, R. M., BOCHNER, B. S., KAGEY-SOBOTKA, A. & LICHTENSTEIN, L. M. 1992. Histaminecontaining cells obtained from the nose hours after antigen challenge have functional and phenotypic characteristics of basophils. J Immunol, 148, 2223-8. JACKSON, D. J., HARTERT, T. V., MARTINEZ, F. D., WEISS, S. T. & FAHY, J. V. 2014. Asthma: NHLBI Workshop on the Primary Prevention of Chronic Lung Diseases. Ann Am Thorac Soc, 11 Suppl 3, S139-45. JEFFERY, P .K., WARDLAW, A. J., NELSON, F. C., COLLINS, J. V. & KAY, A. B. 1989. Bronchial biopsies in asthma. An ultrastructural, quantitative study and correlation with hyperreactivity. Am Rev Respir Dis, 140, 1745-53. JOHN, A. E. & LUKACS, N. W. 2003. Chemokines and asthma. Sarcoidosis Vasc Diffuse Lung Dis, 20, 180-9. JOHNSON, S. R. & KNOX, A. J. 1997. Synthetic functions of airway smooth muscle in asthma. Trends Pharmacol Sci, 18, 288-92. JOSEPH, M., TONNEL, A. B., TORPIER, G., CAPRON, A., ARNOUX, B. & BENVENISTE, J. 1983. Involvement of immunoglobulin E in the secretory processes of alveolar macrophages from asthmatic patients. J Clin Invest, 71, 221-30. 64
KAISER, H. B. 2004. Risk factors in allergy/asthma. Allergy Asthma Proc, 25, 7-10. KALENDERIAN, R., RAJU, L ,.ROTH, W., SCHWARTZ, L. B., GRUBER, B. & JANOFF, A. 1988. Elevated histamine and tryptase levels in smokers' bronchoalveolar lavage fluid. Do lung mast cells contribute to smokers' emphysema? Chest, 94, 119-23. KELLY, C., WARD, C., STENTON, C. S., BIRD ,G., HENDRICK, D. J. & WALTERS, E. H. 1988. Number and activity of inflammatory cells in bronchoalveolar lavage fluid in asthma and their relation to airway responsiveness. Thorax, 43, 684-92. KHARITONOV, S. A., YATES, D., ROBBINS, R. A., LOGAN-SINCLAIR ,R., SHINEBOURNE, E. A. & BARNES, P. J. 1994. Increased nitric oxide in exhaled air of asthmatic patients. Lancet, 343, 133-5. KIMURA, I., TANIZAKI, Y., SAITO, K., TAKAHASHI, K., UEDA, N. & SATO, S. 1975. Appearance of basophils in the sputum of patients with bronchial asthma. Clin Allergy, 5, 95-8. KOMAROW, H. D., HU, Z., BRITTAIN, E., UZZAMAN, A., GASKINS, D. & METCALFE, D. D. 2009. Serum tryptase levels in atopic and nonatopic children. J Allergy Clin Immunol, 124, 845-8. KOSHAK, E. A. 2007. Classification of asthma according to revised 2006 GINA: evolution from severity to control. Ann Thorac Med, 2, 45-6. KRAMME, R. D., HOFFMANN, K.-P. & POZOS, R. S. 2011. Springer handbook of medical technology, Berlin, Springer. KROEGEL, C. 2009. Global Initiative for Asthma (GINA) guidelines: 15 years of application. Expert Rev Clin Immunol, 5, 239-49. KUCHAREWICZ, I., BODZENTA-LUKASZYK, A., SZYMANSKI, W., MROCZKO, B. & SZMITKOWSKI, M. 2007. Basal serum tryptase level correlates with severity of hymenoptera sting and age. J Investig Allergol Clin Immunol, 17, 65-9. LACOSTE, J. Y., BOUSQUET, J., CHANEZ, P., VAN VYVE, T., SIMONYLAFONTAINE, J., LEQUEU, N., VIC, P., ENANDER, I., GODARD, P. & MICHEL, F. B. 1993. Eosinophilic and neutrophilic inflammation in asthma, chronic bronchitis, and chronic obstructive pulmonary disease. J Allergy Clin Immunol, 92, 537-48. LAMBRECHT, B. N., PAUWELS, R. A. & BULLOCK, G. R. 1996. The dendritic cell: its potent role in the respiratory immune response. Cell Biol Int, 20, 111-20.
65
LEE ,R. U. & STEVENSON, D. D. 2011. Aspirin-exacerbated respiratory disease: evaluation and management. Allergy Asthma Immunol Res, 3, 3-10. LEFF, A. R. 2001. Regulation of leukotrienes in the management of asthma: biology and clinical therapy. Annu Rev Med.14-1 ,52 , LEVINE, S. J. 1995. Bronchial epithelial cell-cytokine interactions in airway inflammation. J Investig Med, 43, 241-9. LITTLE, S. S. & JOHNSON, D. A. 1995. Human mast cell tryptase isoforms: separation and examination of substrate-specificity differences. Biochem J, 307 ( Pt 2), 341-6. LIU, T., VALDEZ, R., YOON, P. W., CROCKER, D., MOONESINGHE, R. & KHOURY, M. J. 2009. The association between family history of asthma and the prevalence of asthma among US adults: National Health and Nutrition Examination Survey, 1999-2004. Genet Med, 11, 323-8. LOHI, J., HARVIMA, I. & KESKI-OJA, J. 1992. Pericellular substrates of human mast cell tryptase: 72,000 dalton gelatinase and fibronectin. J Cell Biochem, 50, 337-49. LOMBARDO, L. J. & BALMES, J. R. 2000 .Occupational asthma: a review. Environ Health Perspect, 108 Suppl 4, 697-704. LONGO, D. L. 2012. Harrison's principles of internal medicine, New York, McGraw-Hill. MADDOX, L. & SCHWARTZ, D. A. 2002. The pathophysiology of asthma. Annu Rev Med, 53, 477-98. MAIER, M., SPRAGG, J. & SCHWARTZ, L. B. 1983. Inactivation of human high molecular weight kininogen by human mast cell tryptase. J Immunol, 130, 2352-6. MARUYAMA, N., TAMURA, G., AIZAWA, T., OHRUI, T., SHIMURA, S., SHIRATO, K. & TAKISHIMA, T. 1994. Accumulation of basophils and their chemotactic activity in the airways during natural airway narrowing in asthmatic individuals. Am J Respir Crit Care Med, 150, 1086-93. MASOLI, M., FABIAN, D., HOLT, S., BEASLEY, R. & GLOBAL INITIATIVE FOR ASTHMA, P. 2004. The global burden of asthma: executive summary of the GINA Dissemination Committee report. Allergy, 59, 469-78. MCGRATH, M. E., SPRENGELER, P. A., HIRSCHBEIN, B., SOMOZA, J. R., LEHOUX, I., JANC, J. W., GJERSTAD, E., GRAUPE, M., ESTIARTE, A., 66
VENKATARAMANI ,C., LIU, Y., YEE, R., HO, J. D., GREEN, M. J., LEE, C. S., LIU, L., TAI, V., SPENCER, J., SPERANDIO, D. & KATZ, B. A. 2006. Structure-guided design of peptide-based tryptase inhibitors. Biochemistry, 45, 5964-73. MIELCK, A., REITMEIR, P. & WJST, M. 1996 .Severity of childhood asthma by socioeconomic status. Int J Epidemiol, 25, 388-93. MIN, H. K., KAMBE, N. & SCHWARTZ, L. B. 2001. Human mouse mast cell protease 7-like tryptase genes are pseudogenes. J Allergy Clin Immunol, 107, 315-21. MIN, H. K., MOXLEY ,G., NEALE, M. C. & SCHWARTZ, L. B. 2004. Effect of sex and haplotype on plasma tryptase levels in healthy adults. J Allergy Clin Immunol, 114, 48-51. MURPHY, D. M. & O'BYRNE, P. M. 2010. Recent advances in the pathophysiology of asthma. Chest, 137, 1417.26NAKANISHI, K. 2010. Basophils are potent antigen-presenting cells that selectively induce Th2 cells. Eur J Immunol, 40, 1836-42. NATIONAL ASTHMA, E. & PREVENTION, P. 2007. Expert Panel Report 3 (EPR-3): Guidelines for the Diagnosis and Management of AsthmaSummary Report 2007. J Allergy Clin Immunol, 120, S94-138. O'BYRNE, P. M. 2010. Global guidelines for asthma management: summary of the current status and future challenges. Pol Arch Med Wewn, 120, 511-7. OETTGEN, H. C. & GEHA, R. S. 1999. IgE in asthma and atopy: cellular and molecular connections. J Clin Invest, 104, 829-35. PALLAORO, M., FEJZO, M. S., SHAYESTEH, L., BLOUNT, J. L. & CAUGHEY, G. H. 1999. Characterization of genes encoding known and novel human mast cell tryptases on chromosome 16p .13.3J Biol Chem, 274, 3355-62. PEREIRA, P. J., BERGNER, A., MACEDO-RIBEIRO, S., HUBER, R., MATSCHINER, G., FRITZ, H., SOMMERHOFF, C. P. & BODE, W. 1998. Human beta-tryptase is a ring-like tetramer with active sites facing a central pore. Nature, 392, 30.11-6 RANZI, A., PORTA, D., BADALONI, C., CESARONI, G., LAURIOLA, P., DAVOLI, M. & FORASTIERE, F. 2014. Exposure to air pollution and respiratory symptoms during the first 7 years of life in an Italian birth cohort. Occup Environ Med, 71, 430-6. RATAGERI ,V. H., KABRA, S. K., DWIVEDI, S. N. & SETH, V. 2000. Factors associated with severe asthma. Indian Pediatr, 37, 1072-82. 67
REN, S., SAKAI, K. & SCHWARTZ, L. B. 1998. Regulation of human mast cell beta-tryptase: conversion of inactive monomer to active tetramer at acid pH. J Immunol, 160, 4561-9. RIEDL, M. A. 2008. The effect of air pollution on asthma and allergy. Curr Allergy Asthma Rep, 8, 139-46. ROBINSON, D., HAMID, Q., BENTLEY, A., YING, S., KAY, A. B. & DURHAM, S. R. 1993. Activation of CD4+ T cells ,increased TH2-type cytokine mRNA expression, and eosinophil recruitment in bronchoalveolar lavage after allergen inhalation challenge in patients with atopic asthma. J Allergy Clin Immunol, 92, 313-24. SAETTA, M., DI STEFANO, A., MAESTRELLI, P., DE MARZO ,N., MILANI, G. F., PIVIROTTO, F., MAPP, C. E. & FABBRI, L. M. 1992. Airway mucosal inflammation in occupational asthma induced by toluene diisocyanate. Am Rev Respir Dis, 145, 160-8. SAHINER, U. M., YAVUZ, S. T., BUYUKTIRYAKI, B., CAVKAYTAR, O., YILMAZ ,E. A., TUNCER, A. & SACKESEN, C. 2014. Serum basal tryptase levels in healthy children: correlation between age and gender. Allergy Asthma Proc, 35, 404-8. SAMPSON, A. P. 2000. The role of eosinophils and neutrophils in inflammation. Clin Exp Allergy, 30 Suppl 1, 22-7. SAUNDERS, M. A., MITCHELL, J. A., SELDON, P. M., YACOUB, M. H., BARNES, P. J., GIEMBYCZ, M. A. & BELVISI, M. G. 1997. Release of granulocyte-macrophage colony stimulating factor by human cultured airway smooth muscle cells: suppression by dexamethasone. Br J Pharmacol, 120, 545-6. SCHLIEMANN, S., SEYFARTH, F., HIPLER, U. C. & ELSNER, P. 2012. Impact of age and heterophilic interference on the basal serum tryptase, a risk indication for anaphylaxis, in 1,092 dermatology patients. Acta Derm Venereol, 92, 484-9. SCHWARTZ, L. B. 1992. Cellular inflammation in asthma: neutral proteases of mast cells. Am Rev Respir Dis, 145, S18-21. SCHWARTZ, L. B. 2006. Diagnostic value of tryptase in anaphylaxis and mastocytosis. Immunol Allergy Clin North Am.63-451 ,26 , SCHWARTZ, L. B., BRADFORD, T. R., LITTMAN, B. H. & WINTROUB, B. U. 1985. The fibrinogenolytic activity of purified tryptase from human lung mast cells. J Immunol, 135, 2762-7.
68
SCHWARTZ, L. B., KAWAHARA, M. S., HUGLI, T. E., VIK, D., FEARON, D .T. & AUSTEN, K. F. 1983. Generation of C3a anaphylatoxin from human C3 by human mast cell tryptase. J Immunol, 130, 1891-5. SCHWARTZ, L. B., LEWIS, R. A., SELDIN, D. & AUSTEN, K. F. 1981. Acid hydrolases and tryptase from secretory granules of dispersed human lung mast cells. J Immunol, 126, 1290-4. SCHWARTZ, L. B., MAIER, M. & SPRAGG, J. 1986. Interaction of human low molecular weight kininogen with human mast cell tryptase. Adv Exp Med Biol, 198 Pt A, 105-11. SCHWARTZ, L. B., MIN, H. K., REN, S., XIA ,H. Z., HU, J., ZHAO, W., MOXLEY, G. & FUKUOKA, Y. 2003. Tryptase precursors are preferentially and spontaneously released, whereas mature tryptase is retained by HMC-1 cells, Mono-Mac-6 cells, and human skin-derived mast cells. J Immunol, 170, 5667-73. SCHWARTZ, L. B., YUNGINGER, J. W., MILLER, J., BOKHARI, R. & DULL, D. 1989. Time course of appearance and disappearance of human mast cell tryptase in the circulation after anaphylaxis. J Clin Invest, 83, 1551-5. SELWOOD, T., MCCASLIN, D. R. & SCHECHTER, N .M. 1998. Spontaneous inactivation of human tryptase involves conformational changes consistent with conversion of the active site to a zymogen-like structure. Biochemistry, 37, 13174-83. SHORE, S. A. 2008. Obesity and asthma: possible mechanisms. J Allergy Clin Immunol, 121, 1087-93; quiz 1094-5. SIGURS, N., BJARNASON, R., SIGURBERGSSON, F. & KJELLMAN, B. 2000. Respiratory syncytial virus bronchiolitis in infancy is an important risk factor for asthma and allergy at age 7. Am J Respir Crit Care Med, 161.7-1501 , SMALL-HOWARD, A. & TURNER, H. 2005. Exposure to tobacco-derived materials induces overproduction of secreted proteinases in mast cells. Toxicol Appl Pharmacol, 204, 152-63. SOLE, D. 2013. Obesity and asthma. Rev Paul Pediatr, 31, 136-7. SOMMERHOFF, C. P. 2001. Mast cell tryptases and airway remodeling. Am J Respir Crit Care Med, 164, S52-8. SPORIK, R. & PLATTS-MILLS, T. A. 2001. Allergen exposure and the development of asthma. Thorax, 56 Suppl 2, ii58-63.
69
STACK, M. S. & JOHNSON, D. A. 1994. Human mast cell tryptase activates single-chain urinary-type plasminogen activator (pro-urokinase). J Biol Chem, 269, 9416-9. STAPLETON, M., HOWARD-THOMPSON, A., GEORGE, C., HOOVER, R. M. & SELF, T. H. 2011. Smoking and asthma. J Am Board Fam Med, 24, 313-22. STRACHAN, D. P. 1989. Hay fever, hygiene, and household size. BMJ, 299, 1259-60. SUBBARAO, P., MANDHANE, P. J. & SEARS, M. R. 2009. Asthma: epidemiology, etiology and risk factors. CMAJ, 181, E181-90. SULAKVELIDZE, I., INMAN, M. D., RERECICH, T. & O'BYRNE, P. M. 1998. Increases in airway eosinophils and interleukin-5 with minimal bronchoconstriction during repeated low-dose allergen challenge in atopic asthmatics. Eur Respir J, 11, 821-7. TAMIMI, A., SERDAREVIC, D. & HANANIA, N. A. 2012. The effects of cigarette smoke on airway inflammation in asthma and COPD: therapeutic implications. Respir Med, 106, 319-28. TERAN, L. M. 2000. CCL chemokines and asthma. Immunol Today, 21, 235-42. THOMAS, V. A., WHEELESS, C. J., STACK, M. S. & JOHNSON, D. A. 1998. Human mast cell tryptase fibrinogenolysis: kinetics, anticoagulation mechanism, and cell adhesion disruption. Biochemistry, 37, 2291-8. THOMSON, N. C., CHAUDHURI, R. & LIVINGSTON, E. 2004. Asthma and cigarette smoking. Eur Respir J, 24, 822-33. TOELLE, B. G ,.XUAN, W., PEAT, J. K. & MARKS, G. B. 2004. Childhood factors that predict asthma in young adulthood. Eur Respir J, 23, 66-70. TSUKIOKA, K., TOYABE, S. & AKAZAWA, K. 2010. [Relationship between asthma severity and age at onset in Japanese adults]. Nihon Kokyuki Gakkai Zasshi, 48, 898-905. TZIVIAN, L. 2011. Outdoor air pollution and asthma in children. J Asthma, 48, 470-81. WAHN, U., LAU, S., BERGMANN, R., KULIG, M., FORSTER, J., BERGMANN, K., BAUER, C. P. & GUGGENMOOS-HOLZMANN, I. 1997. Indoor allergen exposure is a risk factor for sensitization during the first three years of life. J Allergy Clin Immunol, 99, 763-9.
70
WALLS, A. F., BRAIN, S. D., DESAI, A., JOSE, P. J., HAWKINGS, E., CHURCH, M. K. & WILLIAMS, T. J. 1992. Human mast cell tryptase attenuates the vasodilator activity of calcitonin gene-related peptide. Biochem Pharmacol, 43, 1243-8. WEIDNER, N. & AUSTEN, K. F. 1990. Evidence for morphologic diversity of human mast cells. An ultrastructural study of mast cells from multiple body sites. Lab Invest, 63, 63-72. WELLE, M. 1997. Development, significance, and heterogeneity of mast cells with particular regard to the mast cell-specific proteases chymase and tryptase. J Leukoc Biol, 61, 233-45. WEN, Y., REID, D. W., ZHANG, D., WARD, C., WOOD-BAKER, R & . WALTERS, E. H. 2010. Assessment of airway inflammation using sputum, BAL, and endobronchial biopsies in current and exsmokers with established COPD. Int J Chron Obstruct Pulmon Dis, 5, 327-34. WILLIAMS, C. M. & GALLI, S. J. 2000. The diverse potential effector and immunoregulatory roles of mast cells in allergic disease. J Allergy Clin Immunol, 105, 847-59. WILLIAMS, T. J. 2004. The eosinophil enigma. J Clin Invest, 113, 507-9. ZHANG, M. Q. & TIMMERMAN, H. 1997. Mast cell tryptase and asthma. Mediators Inflamm, 6, 311-7.
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Appendix Questionnaire Form Case No.: (
)
Name: Sex: ♂ ♀
Age: Mob. No.:
Urban
Residency:
Employment status: Employed Duration of the disease: ( Smoker:
Unemployed
)
Current Social Ex-smoker Never
Family history of Asthma: Yes
No
Daytime symptoms per week: (
)
Limitation of activities:
None
Any
Nocturnal symptoms:
None
Any
Rescue treatment per week: ( PFT (FEV1):
72
)
Rural
الخالصة المقدمة والغرض :الربو هو مرض االلهاب المزمن للمجبري الهنفسية وهصي مب يقبر أكثر من 300مليون شخص في العبلم .الهريبهز ( (Tryptaseهو األنزيم البروهيني السيريني المحبيد وهو الوسيط األكثر وفرة المخزنة في حبيببت الخاليب البدينة .االطالق لهريبهز ( (Tryptaseمن الحبيببت اإلفرازية هو سمة من سمبت هحب الخاليب البدينة .هذه الدراسة هقييم العالقة بين مسهوى هريبهز ) (Tryptaseالمصل لخطورة المرض في المصببين بمرض الربو في محبفظة السليمبنية. طرق العمل :هم سح خمسة ملليليهرات من الدم الوريدي من االشخبص المصببين و الغير المصببين (المجموع = .)145ثم هقسيم االشخبص المصببين بمرض الربو (العدد= )85حس هوجيابت الجينب ( )GINAمن قبل منظمة الصحة العبلمية الى ثالثة مجموعبت المسهجيبين للعالج (العدد = ،)25المسهجيبين بصورة جزئية ( العدد = )30و الغير المسهجيبين (العدد = .)30هم هحليل مسهوى الهريبهز ) (Tryptaseالمصل ببألاليزا (المقبيسة المنبعية المرهبط ببإلنزيم) بطريقة الشطيرة. النتائج :كبن هنبك فرق كبير بين مجموعبت الدراسة لمبدة الهريبهز ،مع أعلى المسهويبت للغير المسهجيبين ( 0،65 ± 7،68نبنوغرام /دل) و أدنى المسهويبت في مجموعة المسهجسبين للعالج ( 0،15 ± 4،4نبنوغرام /دل) .وأظارت النهبئج ارهفبع هريبهز ) (Tryptaseالمصل مع زيبدة العمر .وكبن هنبك فرق كبير بين المدخنين السببقين والغير المدخنين أبدا في المصببين بمرض الربو .لم يهم العثور على فرق ملحوظ في مسهويبت هريبهز ) (Tryptaseالمصل بين الذكور واإلنبث ،وليس هنبك هأثير لوجود أقبر مصببين للربو في العبئلة على مسهوى الهريبهز. األستنتاج :كبن مسهويبت هريبهز ) (Tryptaseالمصل في الربو الخطير والغير المسهجي للعالج أعلى بكثير من المجموعة المسهجيبة و الغيرالمصببين للمرض .وعالوة على ذلك، مسهوى هريبهز ) (Tryptaseالمصل يرهفع مع زيبدة العمر و يكون مرهفعب بين المدخنين السببقين .من نبحية اخرى لم يكن هنبك هأثير كبير للجنس و وجود أقبر مصببين للربو في العبئلة على مسهويبت الهريبهز ).(Tryptase
حكومة إقليم كردستان/العراق وزارة التعليم العالي و البحث العلمي جامعة السليمانية كلية العلوم الطبية كلية الطب عالقة مستويات تريبتز ) (Tryptaseالمصل لشدة المرض في المرضى المصابين بالربو في محافظة السليمانية رسبلة مقدمة الى لجنة الدراسبت العليب في كلية العلوم الطبية /كلية الط السليمبنية كجزء من مهطلببت الحصول على شابدة المبجسهير في علم الكيميبء الحيوي السريري
من قبل
محمد أحمد محمود بكالوريوس الطب العام فرع الكيمياء الحيوي بإشراف األسهبذ الدكهورة
بان موسى رشيد دكتوراه في الكيمياء الحيوي السريري
في جبمعة
پوختە بنچینە وئامانج لە لێکۆڵینەوە: ڕەبۆ نەخۆشی ھەو کردنی درێژخایەنی سیەکانە کە زیاتر لە 300ملیۆن توشبوی لە جیھاندا ھەیە (Tryptase) ..ئەنزیمێکی ھاوتایە کە پرۆتین تێکدەشکێنێت وە زۆرترین ڕێکخەرە کە عەمبار کراوە لە دەنکۆڵەکانی درشتەخانەکان .دەردانی ) (Tryptaseلە دەنکۆڵەکانەوە یەکێکە لە نیشانە تایبەتەکانی رشتنی دەنکۆڵکانی درشتەخانەکان .ئەم لێکۆڵینەوەیە پەیوەندی نێوان ئاستی ئەنزیمی ) (Tryptaseو سەختی نەخۆشی ڕەبۆ لە پارێزگاکەماندا ھەڵدەسەنگێنێت.
ڕێگاکانی
کارکردن:
پێنج مللیلتر لە خوێنی خوێن ھێنەر وەرگیرا لە نەخۆشەکانی ڕەبۆ ،کەسانی تەندروستدا (کۆ ژ .)145=.ھەشتاو پێنج نەخۆشی ڕەبۆ دابەشکران بۆ سێ کۆمەڵە لەسەر ڕێبەری جینا ) (GINAلەالیەن ڕێکخراوی تەندروستی جیھانی کە پێکھاتوون لە ڕەبۆی کۆنترۆڵ کراو (ژمارە= )25وە ڕەبۆی نیمچە کۆنترۆڵ کراو (ژمارە=، )30وە ڕەبۆی کۆنترۆڵ نەکراو(ژمارە= .)30ئاستی ئەنزیمەکە لە رێگەی ئیالیزا شیکاری بۆ کرا.
ئەنجامەکان: ئەنجامەکان دەریدەخەن کە جیاوازییەکی گەورە ھەیە لە نێوان کۆماڵەکانی لێکۆڵینەوەکەد بە شێوەیەک کە بەرزترین ئاستی ئەنزیمی ) (Tryptaseلە ڕەبۆی کۆنترۆڵ نەکراودایە ( 0،65 ± 7،68نگم /دل) وە نزمترین ئاستی ئەنزیمەکە لە ڕەبۆی کۆنترۆل کراودایە ( 0،15 ± 4،4نگم /دل) .ئەنزیمی ) (Tryptaseبە شێوەیەکی بەرچاو زیاد دەکات لەگەڵ زیادبوونی تەمەندا. ھەروەھا جیاوازیەکی بەرچاو ھەیە لە نێوان ئەو کەسانەی کە پێشتر جگەرە کێش بوون و لەگەڵ ئەو کەسانەی کە ھەگیز نەیان کێشاوە .ڕەگەز و بوونی نەخۆشی ڕەبۆ لە ئەندامانی خێزاندا کاریگەری نیە لەسەر ئاستی ئەنزیمی ).(Tryptase
دەرئەنجامەکان :ئاستی ئەنزیمی ) (Tryptaseبە شێوەیەکی بەرچاو
بەرزترە لە ڕەبۆی کۆنترۆڵ نەکراودا بەراورد بە ڕەبۆی کۆنترۆڵ کراو .لەگەڵ ئەوەشدا ئەنزیمی ) (Tryptaseلەگەڵ زیادبوونی تەمەن و ئەو کەسانەی ڕەبۆیان ھەیە و پێشتر جگەرەکێش بوون بەرز دەبێتەوە .لەالیەکی ترەوە ھیچ جیاوازێکی بەرچاو نابینرێ کە ڕەگەز و بوونی ڕەبۆ لە ئەندامانی خێزاندا کاریگەری ھەبێت لەسەر ئاستی ) (Tryptaseلە زەرداوی خوێندا.
حکومهتی ھەرێمی کوردستان/عێراق وهزارهتی خوێندنی بااڵوتوێژینهوهی زانستی زانکۆی سلێمانی فاکهڵتی زانسته پزیشکیەکان سکوڵی پزیشکی
"پەیوەندی ئاستی ئەنزیمی ) (Tryptaseبە سەختی نەخۆشی ڕەبۆ لە توشبوانی نەخۆشێکە لە پارێزگای سلێمانی"
لێکۆڵینهوهکه پێشکهش بە لقى خوێندنی بااڵی سکوڵی پزیشکی /فاکهڵتی زانسته پزیشکی یهکانی زانکۆی سلێمانی کراوه وهک بهشێک لهپێداویستیەکانی بهدهستهێنانی بڕوانامهی ماستهر له زانستی بایۆکیمستری پزیشکی
لەالیەن
محمد احمد محمود بەکالۆریۆس لە پزیشکی گشتی لقی بایۆکیمستری سهرپەرشتیار
دکتۆرە بان موسی رشید دکتۆرا لە بایۆکمستری پزیشکی
